Means and methods for investigating nucleic acid sequences

ABSTRACT

The invention provides improved methods for investigating nucleic acid sequences, wherein at least one additional probe is used which is specific for a (pseudo)gene variant of a target nucleic acid.

The invention relates to the fields of biology, molecular biology,biotechnology and medicine.

Nucleic acid sequences are investigated in a wide variety ofapplications. For instance, for diagnosis of infection with a pathogen,a sample of an individual is often screened for the presence of pathogennucleic acid. Furthermore, nucleic acid sequence investigation is oftenperformed for the diagnosis of genetic disorders, such as for instancePrader-Willi syndrome, Angelman syndrome and Duchenne musculardystrophy. Widely used methods for detection of deletions orduplications of chromosomal sequences are quantitative multiplex PCR andquantitative Southern blotting. Drawbacks of these methods are that theyare time-consuming and that results are difficult to interpret.

One particularly suitable technique for investigation of nucleic acidsequences is multiplex ligation dependent probe amplification (MLPA).This technique is based on hybridisation of probes to target nucleicacids, where after probes are amplified. In currently used MLPA assays,each MLPA probe set consists of two half probes. These two half probescontain a target-specific sequence and a primer binding site sequence towhich a nucleic acid amplification primer (preferably a PCR primer) canbind. One half probe is typically shorter in length then the other. Theother half probe is longer due to a non-hybridizing stuffer sequence.The stuffer sequence of each probe set is unique in length, resulting indifferent lengths of amplification products (typically between 130 and480 base pairs) that can be separated by electrophoresis. In an MLPAassay, typically a plurality of probe sets is used. The two half probesof each probe set are typically added to denatured sample nucleic acidand hybridized immediately adjacent to each other on their targetsequence. Subsequently, the resulting nucleic acid is subjected to aligation reaction. Usually a ligase is used which ligates only halfprobes that are perfectly matched with their target sequence (such asfor instance the thermostable Ligase-65). A mismatch of a half probe atthe ligation site prevents ligation and amplification. Thereby noamplification products of the probe will be detected. This allows MLPAto discriminate sequences that only differ in a single nucleotide.Sequences from pseudogenes or related genes can therefore bedistinguished. Ligated half probes (which are also referred to as“ligated probes”) are amplified, preferably by PCR, using primerscapable of specifically binding the primer binding site sequences of theprobes. The amplification products of each ligated probe are separatedand analyzed, for instance by electrophoresis. Preferably, amplificationproducts are represented graphically by separate peaks. Each peak is theproduct of an amplified MLPA ligated probe and a relative difference inpeak intensity (height or surface) between a control sample and a sampleof interest indicates copy number variation. FIG. 1A schematicallyoutlines an MLPA reaction.

MLPA is particularly suitable for detecting nucleic acid (pseudo)genevariants, (pseudo)gene-specific nucleotides and/or copy numbervariation. MLPA has been employed in several studies, e.g. for thediagnosis of Prader-Willi or Angelman syndromes, for prenatal diagnosisof chromosomal aberrations in fetuses, and for the detection of exondeletions and/or duplications in the Duchenne muscular dystrophy gene.Overall, the conclusion was that MLPA could replace the existing methodsused for screening of chromosomal abnormalities due to its relativesimplicity, reproducibility and speed.

In an MLPA assay, targeted nucleic acid which is gene-specific orpseudogene-specific is preferably present at the ligation site of thehalf probes. When a gene-specific or pseudogene-specific nucleotide ispresent at (or within three nucleotides from) a ligation site, this willensure that only perfectly matched half probes are ligated to eachother. A mismatch of a half probe at the ligation site prevents ligationand amplification, whereas a perfect match of the half probe at theligation site allows ligation and amplification. As said before, thisallows MLPA to discriminate between sequences that only differ in asingle nucleotide. Mismatches at four to six nucleotides away from theligation site have been reported to have little effect on the ligationstep.

Hence, the half probes are preferably designed such that the half probewhose 3′ end hybridizes at a target sequence (called herein a “leftprobe” or a “left half probe”) is complementary to a gene-specificsequence or pseudogene-specific sequence of the target sequence. Thisgene-specific or pseudogene-specific sequence of the target sequencecomprises at least one but preferably more nucleotides that make theprobe specific for a given gene or pseudogene. Preferably, at least oneof the 3′ end nucleotides of said left half probe is complementary to atleast one gene-specific nucleotide and/or at least onepseudogene-specific nucleotide of the target sequence, so that the(pseudo)gene-specific nucleotide(s) or a single nucleotide polymorphismwithin a given (pseudo)gene is present at (or within three nucleotidesfrom) the ligation site of said left half probe. In this case, said lefthalf probe and the probe whose 5′ end hybridizes at a target sequence(called herein a “right probe” or a “right half probe”) are ligated toeach other only when the sequence of the left half probe perfectlymatches its target sequence.

As used herein the term “gene-specific nucleotide” or “gene-specificsequence” means a nucleotide or sequence, respectively, which is presentin said gene but not present at the corresponding location in at leastone other related gene or pseudogene. The term “pseudogene-specificnucleotide” or “pseudogene-specific sequence” means a nucleotide orsequence, respectively, which is present in said pseudogene but notpresent at the corresponding location in at least one other related geneor pseudogene. Hence, at least one other (pseudo)gene comprises anothernucleotide or sequence at that location. The presence of a(pseudo)gene-specific nucleotide or (pseudo)gene-specific sequence in a(pseudo)gene thus distinguishes said (pseudo)gene from at least oneother (pseudo)gene, even in case when the other (pseudo)gene has a highoverall homology with said (pseudo)gene.

A pseudogene is defined herein as a nucleic acid sequence which does notencode a wild type, functional, protein. The term “pseudogene”encompasses nucleic acid sequences which do not encode protein at all.Additionally, the term “pseudogene” encompasses gene alleles whichcomprise a modification, for instance an insertion or deletion so thatthey encode a protein or a part of a protein with significantlyimpaired, or lost, function as compared to a wild type protein of thesame kind. Such allele for instance encodes a truncated protein as aresult of a frame shift caused by an insertion and/or deletion of atleast one nucleotide, or caused by a premature stop codon.

Since ligases only ligate half probes which are adjacent to each other,half probes need to be designed which are capable of hybridizingimmediately adjacent to each other on their target sequence. This is notalways convenient, because the hybridization location of a left halfprobe on a target nucleic acid is often determined by a(pseudo)gene-specific site of the target nucleic acid (as explainedabove). In such case, the sequence of the corresponding right half probeis determined as well, since the right half probe should be capable ofhybridizing to a region of said target nucleic acid which is immediatelyadjacent to said (pseudo)gene-specific nucleotide. However, such regionmay comprise sequences which are very commonly present in the nucleicacid sequences of a sample. As a result, a right half probe having asequence which is complementary to such common sequence will hybridizeat many different sites of the nucleic acids present in a sample. Insuch case, it would be more attractive to design a right half probe witha sequence which is more specific for a given site of interest of atarget nucleic acid. However, if the left half probe and the right halfprobe do not hybridize to adjacent regions of a target nucleic acid, thecommonly used ligases will not be capable of performing the ligationreaction. Patent application WO 01/61033 in the name of Schoutendiscloses a solution to this problem by adding a short third probe tothe reaction mixture, which third probe will fill the gap between theleft half probe and the right half probe. Such third probe is designedto hybridize to a region of a target nucleic acid which lies between theleft and the right half probes. After hybridization of such third probe,the left half probe is connected to the right half probe via the thirdprobe and ligation has become possible. The third half probe does notneed to be perfectly complementary to the region of the target nucleicacid which lies between the left and the right half probes, as long asthe third probe connects the left half probe and the right half probe sothat a ligase reaction can occur. Moreover, since the third probe issmall, it will hybridize more easily to the target nucleic acid ascompared to the left and right half probes. Hence, mismatches betweenthe third probe and the target nucleic acid are allowed. This way, oneand the same third probe is suitable for connecting left and right halfprobes of different probe sets.

Instead of using a third probe, WO 01/61033 also discloses an embodimentwherein the 3′ end of a left half probe is extended after hybridizationof the half probes to the target sequence, so that the gap between theleft half probe and the right half probe is filled. The resultingextended left half probe is adjacent to the right half probe and aligase reaction has become possible.

In order to be capable of distinguishing between amplificates ofdifferent probe sets, currently used MLPA probe sets are designed suchthat the resulting amplificates have a different length. Differences inligated probe length are typically realized by using a non-hybridizingstuffer sequence in one of the half probes. The stuffer sequence of thehalf probes of each probe set is unique in length, resulting indifferent lengths of amplification products that can be separated byelectrophoresis. Typically, in order to be capable of discriminatingbetween the different amplification products, the difference in lengthbetween different ligated probes is at least 5 nucleotides. Since ausual MLPA assay involves the use of many different probe sets in orderto be capable of detecting a wide variety of (pseudo)gene variants, thismeans that long probes have to be generated. This is especially the casewhen complex loci carrying many (pseudo)gene-specific nucleotides areinvestigated for proper genotyping and/or additional single nucleotidepolymorphisms are investigated for detection of subtle genetic variationwithin a specific genotype, as well as the presence of pseudogenes andsingle nucleotides in these pseudogenes. Such investigation requires theuse of many different probe sets. This is inconvenient if probes arechemically synthesized, because a drawback of synthetic probes is thelower quality in comparison with cloned probes, due to contaminationwith incompletely synthesized probes. These incompletely synthesizedprobes lack or gain one nucleotide, which results in stutter peaks andsplit peaks. A method to remove these contaminants is to purify thesynthesized probes, for instance by polyacrylamid gel electrophoresis(PAGE). If short and long probes are chemically synthesized, a higherproportion of longer probes is more likely to be affected by theincomplete oligonucleotides, causing a limitation of synthetic probesize. The upper limit of synthetic probes is typically about 100 basepairs.

On the other hand, the use of synthetic probes is preferred because theyare easy to obtain and cost-effective whereas generating a probe bycloning in bacteriophage vectors is a time-consuming process and moreexpensive.

Hence, although good results have been obtained with currently used MLPAassays, it is desirable to provide alternatives and improvements,especially if complex (pseudo)gene loci are investigated which involvesthe use of many probe sets.

It is an object of the present invention to provide alternative andimproved MLPA methods and MLPA-like methods.

Accordingly, the present invention provides MLPA assays and MLPA-likeassays wherein at least one probe set is used which comprises a firstnucleic acid probe (“left probe” or “left probe part”), a second nucleicacid probe (“right probe” or “right probe part”) and a third nucleicacid probe (“third probe” or “middle probe” or “middle probe part”),wherein at least one third probe is complementary to a target nucleicacid region comprising a (pseudo)gene-specific nucleotide or(pseudo)gene-specific sequence.

The present invention provides a different approach as compared to theprior art. MLPA methods and MLPA-like methods are now provided whereinat least one third probe, but preferably a plurality of third probes, isused in order to detect at least one (pseudo)gene-specific nucleotide ofa target nucleic acid. Hence, an additional probe is used in at leastone of the probe sets, which is specific for a (pseudo)gene-specifictarget nucleic acid. As used herein, an MLPA-like method is defined as amethod comprising the steps of hybridisation of at least two probes to atarget nucleic acid and ligation of at least two probes. Preferably,said MLPA-like method comprises amplification of ligated probes as well.

MLPA methods and MLPA-like methods according to the present inventionhave several advantages as compared to current methods. For instance, ifthe left probe and the third probe of a probe set are both complementaryto target nucleic acid regions comprising (pseudo)gene-specificnucleotides and/or additional single nucleotide polymorphism(s), twodifferent (pseudo)gene-specific target nucleotides or two SNP's or acombination of one (pseudo)gene specific target nucleotide and one SNPare screened using one probe set. It has become possible to use oneprobe set in order to screen for at least two (pseudo)gene variationswhich are located within a region of about 150 nucleotides of a targetnucleic acid. Contrary, in a currently used MLPA assay two separateprobe sets are needed for screening for two variants in a target nucleicacid. This is illustrated by the following example. If a target(pseudo)gene contains a (pseudo)gene variant at location A and atlocation B, an individual may comprise the following alleles: a-b, a-B,A-b and A-B. In order to determine whether allele a-B is present in asample of said individual, a currently used MLPA assay would need aprobe set specific for the “a” and/or “A” (pseudo)gene variant and aprobe set specific for the “B” and/or “b” (pseudo)gene variant. If boththe probe set specific for “a” and the probe set specific for “B”provide a positive result, it is concluded that allele a-B is present insaid individual. With a MLPA method according to the present invention,however, only one probe set is needed wherein the left probe is specificfor the “a” (pseudo)gene variant and the third probe is specific for the“B” (pseudo)gene variant. If an amplification product is obtained, it isimmediately concluded that allele a-B is present in said individual. Ifallele a-B is not present, said probe set according to the inventionwill not yield an amplification product. Hence, it has become possibleto more specifically screen for a given allele.

Moreover, a method of the invention provides an additional advantagewhen two (pseudo)gene variations are located close to each other. If the(pseudo)gene variants at location A and at location B are close to eachother, the use of two different probe sets according to conventionalMLPA techniques is inconvenient or even not possible at all, because thetwo probe sets will hinder each other in view of their close proximity.This will result in less efficient hybridization of the two probe sets,resulting in a lower signal as compared to a method according to theinvention, wherein two (pseudo)gene variants can be detected using onlyone probe set. Hence, a method according to the invention is moresensitive when (pseudo)gene variants are located close to each other (inpractice, this effect will be most profound when the (pseudo)genevariants are located between 20-100 nucleotides from each other). Havingtwo probes to detect a variant at the same position (such as incurrently used MLPA assays) will result in a change in signal intensity,depending on the presence of the (pseudo)gene variant and the binding ofthe probe. The use of more than two probes for one position is notadvised. FIG. 1B schematically outlines an MLPA reaction according tothe invention in which a probe set consisting of three probes is usedfor detecting two SNPs. FIG. 1C shows a non-limiting example of twospecific probe sets according to the invention for detecting two SNPs.

As another example, in case that an individual is heterozygous for theabove mentioned (pseudo)gene, the individual for instance containsalleles a-B and A-b. A conventional MLPA assay would use four probe sets(one specific for “a”, one specific for “A”, one specific for “b” andone specific for “B”). Four positive results would be obtained, becauseall four probe sets would hybridize and result in an amplificationproduct. However, in such case it would still be unknown whether theindividual comprises the alleles a-b and A-B, or the alleles a-B andA-b. With a method according to the present invention, however, it hasbecome possible to directly identify the alleles of said individual. Forinstance, a first probe set of the invention is used comprising a leftprobe specific for “a” and a third probe specific for “b”, together witha second probe set of the invention comprising a left probe specific for“a” and a third probe specific for “B” and a third probe set of theinvention comprising a left probe specific for “A” and a third probespecific for “b” and a fourth probe set of the invention comprising aleft probe specific for “A” and a third probe specific for “B”. Two ofthese probe sets according to the present invention will yield anamplification product, namely the second probe set of the inventioncomprising a left probe specific for “a” and a third probe specific for“B” and the third probe set of the invention comprising a left probespecific for “A” and a third probe specific for “b”. The first andfourth probe sets according to the present invention will not yield(significant) amplification product. This way, it is immediatelyapparent which alleles are present in said individual. This, too, is anadvantage as compared to currently used methods, especially when complexloci with many (pseudo)gene-specific nucleotides and additional singlenucleotide polymorphisms within a given (pseudo)gene are investigated,because in such case many different combinations of such (pseudo)genevariants need to be screened for.

Another advantage of a method according to the present invention is thefact that more variations in length of the ligated probes are obtained.Since at least one probe set of the invention, but preferably aplurality of probe sets of the invention, comprise a third probe it hasbecome possible to design the probe sets such that variations in lengthof the resulting ligated probes are obtained. This obviates the need ofstuffer sequences. As a result, the individual probes of a probe setaccording to the invention can be kept shorter, which is particularlyadvantageous when chemically synthesized probes are used becausechemical production of long probes is cumbersome, as explained above.Hence, a method according to the invention allows for the use of probesets with relatively short probes, while the resulting ligated probesare long enough to allow for many size variations. Thus, the presentinvention allows the use of synthetic probes, which are easy to obtainand cost-effective, even when complex loci are investigated, and offersgreater flexibility to adapt the assay in case of cross-reactivity orunclear results.

For instance, if 20 (pseudo)gene variants are investigated, probes witha stuffer sequence with a length varying from 4 to 100 nucleotides wouldneed to be used in a conventional MLPA assay in order to be capable ofdistinguishing the resulting amplification products by size. Since theprobe sequences hybridizing to a target sequence are typically about 30nucleotides, and since the primer binding sequences of the probes aretypically about 15-25 nucleotides, this would mean that probe sets withprobes with a length varying from 45-125 nucleotides would need to besynthesized. When the probes are chemically synthesized, it is hardlypossible to obtain reliable probe sets with these lengths. With a methodaccording to the invention, however, differences of length between thevarious amplificates need not to be obtained by use of stuffer sequencesin the probe sets. Instead, at least one third probe is used, preferablya plurality of third probes is used. By varying combinations of threeprobes, optionally in combination with probe sets consisting of twoprobes, the overall length differences of the ligated probes varyconsiderably whereas probe sets can be used with chemically synthesizedprobes with convenient lengths. Of course, this does not mean that theuse of stuffer sequences is excluded. But the skilled person does nolonger have to rely on these stuffer sequences only for lengthvariations. If stuffer sequences are used in a method according to theinvention, it is preferred to keep these sequences as short as possible.

Accordingly, the present invention provides a method for screening forthe presence of at least one target nucleic acid sequence in a sample,comprising the steps of:

-   -   a) adding to said sample at least two different probe sets, each        probe set comprising:        -   a first nucleic acid probe (“left probe”), said first probe            comprising a first nucleic acid sequence complementary to a            first region of said target nucleic acid sequence, and        -   a second nucleic acid probe (“right probe”), said second            probe comprising a second nucleic acid sequence            complementary to a second region of said target nucleic acid            sequence,    -   wherein at least one of said probe sets comprises a third        nucleic acid probe, said third probe comprising a third nucleic        acid sequence complementary to a third region of said target        nucleic acid sequence, and    -   wherein, if said third probe is present in said probe set, said        first and said third region of said target nucleic acid are        located essentially adjacent to each other and said third and        said second region of said target nucleic acid are located        essentially adjacent to each other, and    -   wherein, if said third probe is not present in said probe set,        said first and said second region of said target nucleic acid        are located essentially adjacent to each other,    -   b) allowing hybridization of said at least two different probe        sets to complementary nucleic acid of said sample,    -   c) subjecting nucleic acid of said sample to a ligation        reaction, and    -   d) determining whether said at least one target nucleic acid        sequence is present in said sample,        wherein at least one third nucleic acid probe is complementary        to a target nucleic acid region comprising a (pseudo)gene        variation.

The advantage of probe sets comprising at least three probes accordingto the present invention is that at least two different SNPs can bedetected with one probe set. For instance, in a probe set comprisingthree probes two sites for ligation are present. A left probe and middleprobe are ligated, and a middle probe and right probe are ligated. Ateach ligation site a SNP can be detected. Thus it is possible to designtwo probes of the same probe set in such a way that they are used todetect two SNPs. In that case, using MLPA and a probe set comprisingthree probes according to the invention, a product will only be obtainedwhen both SNPs are present in a sample, because only then ligation canoccur at both ligation sites.

With conventional MLPA probesets consisting of two probes only one SNPcan be detected, because only one site for ligation is present.Additional third probe parts in conventional MLPA, as described in WO01/61033, are occasionally used to bridge the two half probes. Such anadditional third probe part is not SNP-specific. Therefore, theadvantages of probe sets comprising at least three probes according tothe present invention are not obtained when using such additional thirdprobe part for bridging purposes in conventional MLPA.

Therefore, in a preferred embodiment of the invention a probe setcomprises three nucleic acid probes wherein each of at least two nucleicacid probes are specific for a different (pseudo)gene variation.Preferably, a first (or a second) nucleic acid probe of a probe setaccording to the invention is complementary to a target nucleic acidregion comprising a gene-specific nucleotide and/or apseudogene-specific nucleotide and/or a gene-specific sequence and/or apseudogene-specific sequence and/or a polymorphism within a given geneor pseudogene, and a third nucleic acid probe of the same probeset iscomplementary to another target nucleic acid region comprising agene-specific nucleotide and/or a pseudogene-specific nucleotide and/ora gene-specific sequence and/or a pseudogene-specific sequence and/or apolymorphism within a given gene or pseudogene. Said polymorphismpreferably comprises an SNP.

Preferably, ligated probes are amplified. Accordingly, the presentinvention provides a method for screening for the presence of at leastone target nucleic acid sequence in a sample, comprising the steps of:

-   -   a) adding to said sample at least two different probe sets, each        probe set comprising:        -   a first nucleic acid probe (“left probe”), said first probe            comprising a first nucleic acid sequence complementary to a            first region of said target nucleic acid sequence and,            located 5′ thereof, a non-complementary nucleic acid            sequence comprising a first primer binding site, and        -   a second nucleic acid probe (“right probe”), said second            probe comprising a second nucleic acid sequence            complementary to a second region of said target nucleic acid            sequence and, located 3′ thereof, a non-complementary            nucleic acid sequence comprising a second primer binding            site,    -   wherein at least one of said probe sets comprises a third        nucleic acid probe, said third probe comprising a third nucleic        acid sequence complementary to a third region of said target        nucleic acid sequence, and    -   wherein, if said third probe is present in said probe set, said        first and said third region of said target nucleic acid are        located essentially adjacent to each other and said third and        said second region of said target nucleic acid are located        essentially adjacent to each other, and    -   wherein, if said third probe is not present in said probe set,        said first and said second region of said target nucleic acid        are located essentially adjacent to each other,    -   b) allowing hybridization of said at least two different probe        sets to complementary nucleic acid of said sample,    -   c) subjecting nucleic acid of said sample to a ligation        reaction,    -   d) subjecting nucleic acid of said sample to a nucleic acid        amplification reaction, using at least one primer capable of        specifically binding said first primer binding site and at least        one primer capable of specifically binding said second primer        binding site, and    -   e) determining whether amplified nucleic acid is present,        thereby determining whether said at least one target nucleic        acid sequence is present in said sample,    -   wherein at least one third nucleic acid probe is complementary        to a target nucleic acid region comprising a (pseudo)gene        variation.

As used herein, the term “(pseudo)gene variation” encompasses a(pseudo)gene-specific nucleotide and/or a (pseudo)gene-specificsequence. In one embodiment, said (pseudo)gene variation comprises anadditional polymorphism within a given (pseudo)gene. Said additionalpolymorphism preferably comprises an SNP.

Hence, the present invention uses probe sets, wherein at least one probeset, but preferably a plurality of probe sets, comprises three probes.The probes comprise sequences which are complementary to a region of atarget nucleic acid of interest. As used herein, the term“complementary” means that said probe sequence comprises at least 70%,preferably at least 80%, more preferably at least 85%, more preferablyat least 90%, most preferably at least 95% sequence identity to saidregion or to the complement of said region. The term “% sequenceidentity” is defined herein as the percentage of residues in anucleotide sequence that is identical with the residues in a referencesequence after aligning the two sequences and introducing gaps, ifnecessary, to achieve the maximum percent identity. Methods and computerprograms for the alignment are well known in the art. One computerprogram which may be used or adapted for purposes of determining whethera candidate sequence falls within this definition is Autoassembler 2.0(ABI Prism, Perkin Elmer).

The first and second probes of each probe set also comprise a primerbinding site, so that the resulting ligated probes can be amplified.Preferably, the primer binding sites of the first nucleic acid probes ofeach probe set is designed such that the same primer can bind. Thisallows the use of the same primer for binding the primer binding sitesof the first probes in step d). Likewise, it is preferred that theprimer binding sites of the second nucleic acid probes of each probe setis designed such that the same primer can bind. Most preferably, theprobe sets are designed such that a first primer is capable ofspecifically binding the primer binding sites of the first nucleic acidprobes of each probe set and a second primer is capable of specificallybinding the primer binding sites of the second nucleic acid probes ofeach probe set. This embodiment allows the use of only one primer pairin step d). This is, however, not necessary: it is also possible to usedifferent primers for different probe sets. The number of differentprimers is, however, kept as low as possible.

One preferred embodiment therefore provides a method according to theinvention, wherein the first primer binding sites of the first nucleicacid probes of each probe set is capable of specifically binding thesame primer and/or wherein the second primer binding sites of the secondnucleic acid probes of each probe set is capable of specifically bindingthe same primer. Preferably, the first nucleic acid probes and/or thesecond nucleic acid probes of each probe set comprise essentiallyidentical primer binding sequences. Further provided is therefore amethod according to the invention, wherein the non-complementary nucleicacid sequences of said first nucleic acid probes comprise essentiallyidentical first primer binding sites and/or wherein thenon-complementary nucleic acid sequences of said second nucleic acidprobes comprise essentially identical second primer binding sites. Usingessentially identical primer binding sequences ensures that the sameprimer can bind different probes. The term “essentially identical primerbinding sequences” is defined herein as primer binding sequences whichcomprise at least 80%, preferably at least 85%, more preferably at least90%, most preferably at least 95% sequence identity to each other.

As already described, a method according to the invention isparticularly suitable for investigating a nucleic acid sequence havingvarious (pseudo)gene specific nucleotides and/or (pseudo)gene variants,such as complex loci. It is therefore preferred to use a plurality ofthird probes, so that many (pseudo)gene variant combinations areinvestigated. A method according to the invention is thereforepreferably provided wherein at least two, preferably at least five, morepreferably at least ten different third nucleic acid probes are used. Asillustrated in the Examples, a plurality of probe sets comprisingdifferent third probes according to the invention allows for screeningof complex gene loci such as the KIR locus. Not all third probes need tobe specific for a genetic variation of a target nucleic acid. It is alsopossible to use a combination of variant-specific third probes and thirdprobes which are not specific for a (pseudo)gene variation. Likewise,not all first probes need to be specific for a variant of a targetnucleic acid. It is also possible to use a combination ofvariant-specific first probes and first probes which are not specificfor a (pseudo)gene variation. Any of these combinations is for instanceused to vary the length of the resulting ligated probes to a largerextent. In one preferred embodiment of the invention, therefore, atleast 50%, preferably at least 70%, more preferably at least 80%, mostpreferably at least 90% of the third nucleic acid probes iscomplementary to a target nucleic acid region comprising a (pseudo)genevariation. In one embodiment, all third probes are complementary to atarget nucleic acid region comprising a (pseudo)gene variant.Preferably, the second probes (“right probes”) are not designed tocontain (pseudo)gene variant-specific sequences, although the use ofvariant-specific right probes in a method according to the invention isnot excluded.

Preferably, at least 50%, preferably at least 70%, more preferably atleast 80%, most preferably at least 90% of the third nucleic acid probesthat are complementary to a target nucleic acid region comprising a(pseudo)gene variation are combined with a first nucleic acid probe or asecond nucleic acid probe that is complementary to another targetnucleic acid region comprising a (pseudo)gene variation in order to becapable of screening for many variants with one MLPA assay or MLPA-likeassay. In one embodiment, all third probes that are combined with afirst nucleic acid probe or a second nucleic acid probe that iscomplementary to a target nucleic acid region comprising a (pseudo)genevariation are complementary to a target nucleic acid region comprising a(pseudo)gene variant. Of course, these probes are preferably specificfor different variants.

In one preferred embodiment, a (pseudo)gene variant-specific sequence ofa third probe is at least located within the last three nucleotides orthe first three nucleotides of the third probe. This means that the lastthree nucleotides and/or the first three nucleotides comprise at leastone nucleotide which is specific for a (pseudo)gene variation of atarget nucleic acid. In this embodiment, said (pseudo)gene variation ispresent at a ligation site of the third probe, so that ligation is onlypossible when the sequence of the third probe is exactly complementaryto said (pseudo)gene variation. This enhances the specificity of theMLPA method, as explained before. Preferably, the last three nucleotidesand/or the first three nucleotides of said third probe comprise onenucleotide which is specific for a (pseudo)gene variant of a targetnucleotide.

The probe sets according to the present invention preferably have alength between 90 and 300 nucleotides. Cloned probes can be as long as500 nucleotides. Preferably, however, chemically synthesized probes areused because they are rapidly synthesized, easy to obtain andcost-effective. In order to be capable of synthetically producing theprobes according to the present invention, a method according to theinvention is preferably provided wherein third nucleic acid probes witha length of between 20 and 100 nucleotides are used. Most preferably,third nucleic acid probes with a length of between 19 and 110nucleotides are used. Since at least one probe set of the invention, butpreferably a plurality of probe sets according to the invention, is usedwhich comprise three nucleic acid probes, sufficient variations inlength and specificity of the resulting ligated probes is ensured sothat many (pseudo)gene variations can be investigated simultaneously.

These length variations of the resulting ligated probes obviate the needof stuffer sequences, as explained before. It is therefore possible todesign the probe sets such that the parts of the first and/or secondprobe which are not complementary to a target nucleic acid have aboutthe same length. According to this embodiment, the length of thenon-complementary sequences of all first probes is about the same ineach probe set, and/or the length of the non-complementary sequences ofall second probes is about the same in each probe set. These lengths areabout the same when they do not differ from each other by more than 10nucleotides. Preferably, they do not differ from each other by more than6 nucleotides, most preferably they do not differ from each other bymore than 4 nucleotides. This, too, facilitates synthetic production ofthe probes. Further provided is therefore a method according to theinvention, wherein the difference in length of said non-complementarynucleic acid sequences of said first nucleic acid probes of said atleast two different probe sets and/or the difference in length of saidnon-complementary nucleic acid sequences of said second nucleic acidprobes of said at least two different probe sets is less than 6,preferably less than 4 nucleic acids.

Besides the analysis of (pseudo)gene-specific nucleotides and additionalsingle nucleotide polymorphisms, an MLPA technique or MLPA-liketechnique is particularly suitable for relative (pseudo)gene copy numberdetermination. If multiple copies of a (pseudo)gene of interest (or anyother target nucleic acid of interest) are present in sample nucleicacid molecules, each copy will, in principle, be bound by the specificprobes which is detectable. When the probes are amplified, moreamplification product will be present when multiple copies were presentin the original sample nucleic acid as compared to a situation whereinonly one copy is present. Analysis of the amount of amplificationproduct thus provides information about the copy number of a targetnucleic acid of interest. This is often done by graphically representingamplified products by separate peaks. Each peak is the product of anamplified MLPA ligated probe and a relative difference in peak intensity(height or surface) between a control sample and a sample of interestindicates copy number variation. When a complex locus is investigated,multiple copies of a (pseudo)gene of interest can be present in highlypolymorphic regions. In such case, when (pseudo)gene copy number is tobe determined, many different combinations of (pseudo)gene variants needto be taken into account. This involves the use of a wide variety ofdifferent probe sets, to ensure that each combination of (pseudo)genevariants can be detected. In one embodiment according to the presentinvention, however, when the relative copy number of a nucleic acid ofinterest is to be estimated, an improved approach is provided. Accordingto this embodiment, at least one probe is used with degenerate bases atone or more positions. This means that a mixture of probes is usedwherein different nucleotides can be present at one or more positions.Hence a mixture of probes is used, which probes have the same sequence,except for the fact that some probes have a certain nucleotide at agiven position X and some probes have another nucleotide at saidposition X. Such degenerate bases are commonly represented by the IUBnucleotide codes as depicted in FIG. 2. The use of probes withdegenerate bases allows for an efficient estimation of copy number of anucleic acid of interest, even in highly polymorphic regions. Furtherprovided is therefore a method for determining the copy number of anucleic acid of interest, wherein at least one probe set is used whichcomprises a probe with (a) degenerate base(s) at one or more positions.Preferably, at most 20 probe positions have such multiple alternatives,in order to retain specificity of the probes for a given target regionof interest. A use of at least one probe set for determining the copynumber of a nucleic acid of interest, wherein at least one probe setcomprises a probe with (a) degenerate base(s) at one or more positions,is also provided herewith. In one preferred embodiment, at least oneprobe set comprising a probe with (a) degenerate base(s) is used in aMLPA method or MLPA-like method according to the present invention.Further provided is therefore a method according to the invention,wherein at least one probe set is used which comprises a probe with (a)degenerate base(s) at one or more positions.

Alternatively, or additionally, a probe set is used which comprises analternative base which alternative base is capable of binding at leasttwo bases selected from the group consisting of A, T, G, C and U.Preferably, said alternative base is capable of binding at least three,most preferably at least four, bases selected from the group consistingof A, T, G, C and U. Such alternative base is suitable as an alternativefor degenerate bases. It is, of course, also possible to combine suchalternative base with degenerate bases. In a particularly preferredembodiment said alternative base is deoxyinosine triphosphate (dITP) ora functional equivalent thereof, which is capable of binding A and T andG and C and U. Further provided is therefore a method for determiningthe copy number of a nucleic acid of interest, wherein at least oneprobe set is used which comprises an alternative base which is capableof binding at least two, preferably at least three, more preferably atleast four bases selected from the group consisting of A, T, G, C and U.As said before, said alternative base preferably comprises deoxyinosinetriphosphate (dITP) or a functional equivalent thereof. A use of atleast one probe set for determining the copy number of a nucleic acid ofinterest, wherein at least one probe set comprises an alternative basewhich is capable of binding at least two, preferably at least three,more preferably at least four bases selected from the group consistingof A, T, G, C and U, is also provided herewith. In one preferredembodiment, at least one probe set comprising such alternative base(s)is used in a MLPA method or MLPA-like method according to the presentinvention. Further provided is therefore a method according to theinvention, wherein at least one probe set is used which comprises analternative base which is capable of binding at least two, preferably atleast three, more preferably at least four bases selected from the groupconsisting of A, T, G, C and U. As said before, said alternative basepreferably comprises deoxyinosine triphosphate (dITP) or a functionalequivalent thereof.

The present invention provides alternative and improved methods forscreening for the presence of at least one target nucleic acid sequencein a sample, wherein at least one third probe is used which iscomplementary to a target nucleic acid region comprising a (pseudo)genevariation. A use of a probe set comprising at least three nucleic acidprobes, wherein at least one third probe is complementary to a targetnucleic acid region comprising a gene variant and/or a pseudogenevariant, for screening for the presence of at least one target nucleicacid sequence in a sample is therefore also provided. Preferably, aplurality of probe sets according to the present invention is used.Further provided is therefore a use of a plurality of probe sets forscreening for the presence of at least one target nucleic acid sequencein a sample, wherein each of said probe sets comprises:

-   -   a first nucleic acid probe, said first probe comprising        -   a first nucleic acid sequence complementary to a first            region of said target nucleic acid sequence and, located 5′            thereof, a non-complementary nucleic acid sequence            comprising a first primer binding site, and    -   a second nucleic acid probe, said second probe comprising        -   a second nucleic acid sequence complementary to a second            region of said target nucleic acid sequence and, located 3′            thereof, a non-complementary nucleic acid sequence            comprising a second primer binding site,    -   wherein at least one of said probe sets comprises a third        nucleic acid probe, said third probe comprising a third nucleic        acid sequence complementary to a third region of said target        nucleic acid sequence, and    -   wherein, if said third probe is present in said probe set, said        first and said third region of said target nucleic acid are        located essentially adjacent to each other and said third and        said second region of said target nucleic acid are located        essentially adjacent to each other, and    -   wherein, if said third probe is not present in said probe set,        said first and said second region of said target nucleic acid        are located essentially adjacent to each other, and    -   wherein at least one third nucleic acid probe is complementary        to a target nucleic acid region comprising a gene-specific        nucleotide and/or a pseudogene-specific nucleotide and/or a        gene-specific sequence and/or a pseudogene-specific sequence        and/or an additional polymorphism within a given gene or        pseudogene, said polymorphism preferably comprising an SNP.

A method according to the present invention is particularly suitable foranalysis of (pseudo)gene variation and (pseudo)gene copy numberdetermination in complex loci such as the gene encoding complementfactors (e.g. Factor H and FH-like genes, C4A and C4B within theHLA-class III region), chemokines and their receptor alleles (e.g.CCL3L1, CCL4L1, CCR5 or CCR5delta32), HLA-class I and II, SIRPs andLILRs.

In one preferred embodiment, a method according to the invention is usedin order to investigate the killer cell immunoglobulin-like receptor(KIR) locus. KIRs are expressed by natural killer (NK) cells and asubset of T cells. NK cells are cells of the lymphoid lineage, butdisplay no antigen-specific receptors. Their main function is to monitorhost cells for the presence of MHC class I molecules and this isimportant for e.g. distinguishing healthy cells from virus-infected ortumors cells. Interaction between NK cells and MHC class I molecules ismediated by KIRs. The KIR locus in humans is polygenic and highlypolymorphic, so that accurate and efficient characterization of anindividual's KIR (pseudo)gene profile is cumbersome. In thedetermination of the KIR (pseudo)gene profile and their role in manydiseases an efficient and reliable method for KIR genotyping is,however, important. Until now, KIR genotyping is based upon thepolymerase chain reaction sequence-specific primer (PCR-SSP) (Sun et al,2004), multiplex PCR (Vilches et al, 2007) and PCR-sequence specificoligonucleotide probes (PCR-SSOP) (Crum et al, 2000). For the PCR-SSPhigh-quality genomic DNA is required and multiple reactions are neededto generate a complete KIR profile of an individual. Multiple copies ofKIR2DL4 and KIR3DL1/S1 in individuals have been reported with PCR-SSOP(Williams et al, 2003). Detection of the multiple gene copies waspossible because the gene copies of these genes consisted of differentalleles. However, multiple gene copies of highly homologous or identicalsequences are not distinguishable with this molecular detection systemor cloning methods when individuals are homozygous for a gene (Williamset al, 2003).

As shown in the Examples, a method according to the present invention isparticularly suitable for investigating the KIR locus of individuals.Even though this locus is highly polymorphic, (pseudo)gene variants andcopy number variations are efficiently detected with methods accordingto the present invention. One preferred embodiment therefore provides amethod or use according to the invention, wherein said target nucleicacid sequence is present in a KIR locus. Preferably, copy numbervariation of at least one KIR gene and/or at least one KIR pseudogene isdetermined. FIGS. 3A and B provides KIR-specific probes which provideparticularly good results. These probes are therefore preferred when aKIR locus is investigated. FIGS. 3C and D provides an extended list ofKIR-specific probes which provide even better results than the probeslisted in FIGS. 3A and B. Therefore, these probes are even morepreferred when a KIR locus is investigated. Further provided is thus amethod and/or a use according to the invention, wherein at least oneprobe depicted in FIG. 3A, 3B, 3C or 3D, preferably in FIG. 3C or 3D, isused. Preferably, at least two probes depicted in FIG. 3 are used. Inanother preferred embodiment at least four probes, more preferably atleast six probes depicted in FIG. 3A, 3B, 3C or 3D are used.

In a particularly preferred embodiment, a probe set of FIG. 3 is used.Said probe set preferably comprises three probes. A probe set of FIG. 3is formed by two or three individual probes depicted in FIG. 3 whichhave the same number, followed by the letter A, B, C, D, E, G, K, L, Mor N. For instance, probe set 408 is formed by probes 408A, 408B and408C. Optionally, four different probes with the same number are givenfor a probe set of FIG. 3. In that case, a left, a middle and a rightprobe is selected from said four probes. Further provided is therefore amethod and/or a use according to the invention, wherein at least oneprobe set depicted in FIG. 3A selected from the group consisting ofprobe set 408, probe set 507, probe set 419, probe set 528, probe set413, probe set 416, probe set 415 and probe set 418 is used. In aparticularly preferred embodiment at least one probe set depicted inFIG. 3A selected from the group consisting of probe set 408, probe set507, probe set 528, probe set 413, probe set 416 and probe set 415 isused. These probe sets contain a third probe which is specific for a(pseudo)gene variant of the KIR locus. Also provided is a method and/ora use according to the invention, wherein at least one probe setdepicted in FIG. 3B selected from the group consisting of probe set 409,probe set 506, probe set 507, probe set 538, probe set 417 and probe set517 is used. In a particularly preferred embodiment at least one probeset depicted in FIG. 3B selected from the group consisting of probe set409, probe set 506, probe set 507, probe set 538, probe set 417 andprobe set 517 is used. These probe sets also contain a third probe whichis specific for a (pseudo)gene variant of the KIR locus. Also providedis a method and/or a use according to the invention, wherein at leastone probe set depicted in FIG. 3C selected from the group consisting ofprobe set 415, probe set 703, probe set 413, probe set 419, probe set702, probe set 711, probe set 408, probe set 507, probe set 710, probeset 528, probe set 418 and probe set 416 is used. In a particularlypreferred embodiment at least one probe set depicted in FIG. 3C selectedfrom the group consisting of probe set 415, probe set 703, probe set413, probe set 419, probe set 702, probe set 711, probe set 408, probeset 507, probe set 710, probe set 528, probe set 418 and probe set 416is used. These probe sets also contain a third probe which is specificfor a (pseudo)gene variant of the KIR locus. Also provided is a methodand/or a use according to the invention, wherein at least one probe setdepicted in FIG. 3D selected from the group consisting of probe set 506,probe set 417, probe set 517, probe set 409, probe set 507, probe set710, probe set 709, probeset 708, probe set 704 and probe set 538 isused. In a particularly preferred embodiment at least one probe setdepicted in FIG. 3D selected from the group consisting of probe set 506,probe set 417, probe set 517, probe set 409, probe set 507, probe set710, probe set 709, probeset 708, probe set 704 and probe set 538 isused. These probe sets also contain a third probe which is specific fora (pseudo)gene variant of the KIR locus.

It is preferred to use at least two probe sets selected from FIG. 3, sothat various KIR (pseudo)gene variants are screened for with goodresults. More preferably, at least three probe sets selected from FIG. 3are used. Even more preferably, at least four, more preferably at leastfive, most preferably at least six probe sets selected from FIG. 3 areused. Said at least two, three, four, five or six probe sets arepreferably selected from the group consisting of probe set 408, probeset 507, probe set 528, probe set 413, probe set 416, probe set 415,probe set 418, probe set 419, probe set 409, probe set 506, probe set538, probe set 417, probe set 517, probe set 703, probe set 702, probeset 711, probe set 710, probe set 709 and probe set 704 since theseprobe sets contain a third probe which is specific for a (pseudo)genevariant of the KIR locus. In one embodiment, all probe sets depicted inFIG. 3A, and/or 3B, and/or 3C, and/or 3D are used. In a preferredembodiment all probe sets depicted in FIG. 3C and/or FIG. 3D are used.

It is of course also possible to modify a sequence of at least one probedepicted in FIG. 3 to some extent. This is for instance done foroptimalization purposes. Further provided is therefore a method and/or ause according to the invention, wherein at least one probe is used whichhas at least 70%, preferably at least 80%, more preferably at least 85%,more preferably at least 90%, most preferably at least 95% sequenceidentity to a probe depicted in FIG. 3. Preferably, at least two, morepreferably at least four, most preferably at least six probes are usedwhich have at least 70%, preferably at least 80%, more preferably atleast 85%, more preferably at least 90%, most preferably at least 95%sequence identity to a probe depicted in FIG. 3. In one embodiment, amethod or use according to the invention is provided wherein at least 20probes are used, said at least 20 probes having at least 70%, preferablyat least 80%, more preferably at least 85%, more preferably at least90%, most preferably at least 95% sequence identity to the probesdepicted in FIG. 3. A minimum of two specific probes per (pseudo)gene ispreferred to determine copy number variation (CNV).

Preferably, probe sets are used which are based on the probe setsdepicted in FIG. 3A, 3B, 3C or 3D, preferably based on the probe setsdepicted in FIG. 3C and/or 3D. Said probe set preferably comprises threeprobes. One or more of the probes of such probe set may be modified tosome extent, as described above. Further provided is therefore a methodand/or a use according to the invention, wherein at least one probe setis used which has at least 70%, preferably at least 80%, more preferablyat least 85%, more preferably at least 90%, most preferably at least 95%sequence identity to a probe set as depicted in FIG. 3. This means thatthe probes of said probe set have at least 70% sequence identity to thecorresponding probes of at least one probe set of FIG. 3. Preferably, aprobe set is used which has at least 70%, preferably at least 80%, morepreferably at least 85%, more preferably at least 90%, most preferablyat least 95% sequence identity to a probe set depicted in FIG. 3selected from the group consisting of probe set 408, probe set 507,probe set 419, probe set 528, probe set 413, probe set 416, probe set415, probe set 418, probe set 419, probe set 409, probe set 506, probeset 538, probe set 417, probe set 517, probe set 703, probe set 702,probe set 711, probe set 710, probe set 709 and probe set 704 sincethese probe sets contain a third probe specific for a KIR nucleic acidsequence. Preferably at least two, more preferably at least three, morepreferably at least four, more preferably at least five, most preferablyat least six of such probe sets are used, so that various KIR(pseudo)gene variants are screened for with good results.

Novel probes and probe sets which are particularly suitable for(pseudo)gene variant analysis and (pseudo)gene copy number determinationof the KIR locus are also provided. These probes and probe sets arelisted in FIG. 3A, B, C and D, as described above. Further provided aretherefore probes and probe sets as depicted in FIG. 3A, 3B, 3C or 3D, aswell as probes and probe sets which have at least 70%, preferably atleast 80%, more preferably at least 85%, more preferably at least 90%,most preferably at least 95% sequence identity to a probe or probe setdepicted in FIG. 3A, 3B, 3C or 3D. A mixture of nucleic acids, whereinsaid nucleic acids comprise at least two probe sets according to theinvention is also provided. Preferably, said mixture comprises at leastfour, more preferably at least six probe sets according to theinvention. As said before, such probe sets have at least 70% sequenceidentity to a probe or probe set depicted in FIG. 3A, 3B, 3C or 3D. Oneembodiment provides a mixture of nucleic acids comprising at least two,preferably at least four, more preferably at least six probe sets asdepicted in FIG. 3A, 3B, 3C or 3D.

Further provided is a kit for detecting the presence of at least onetarget nucleic acid sequence in a sample, comprising a probe set or amixture of nucleic acids according to the invention. Said at least onetarget nucleic acid sequence preferably comprises a nucleic acidsequence present in a KIR locus. A kit according to the inventionpreferably further comprises a PCR primer set comprising at least 70%,preferably at least 80%, more preferably at least 85%, more preferablyat least 90%, most preferably at least 95% sequence identity to nucleicacid sequences 5′-GGGTTCCCTAAGGGTTGGA and TCTAGATTGGATCTTGCTGGCAC-3′, orthe complements thereof. These primers are particularly suitable foramplifying probe sets depicted in FIG. 3.

KIR polymorphisms have been associated with disease. Association betweenKIR polymorphisms and subtypes of leukemia were investigated by Zhang etal. (Zhang et al. 2009). The presence of KIR2DS4 was demonstrated to bepredisposing to chronic myelogenous leukemia (CML) and the absence ofKIR2DS3 was predisposing to acute lymphoblastic leukemia (ALL). KIR2DS4is present in haplotype A, whereas KIR2DS3 is present in haplotype B.Presence of KIR2DS4 and absence of KIR2DS3 are predisposing to leukemiasubtypes. Thus, characteristics of haplotype A are predisposing toleukemia subtypes. The present invention provides probes that areparticularly well suitable for detecting KIR genes, including KIR2DS4and KIR2DS3. Thus, with probes according to the present inventionselected from FIG. 3A, 3B, 3C and/or 3D the presence and/or absence ofKIR2DS4 and KIR2DS3 in a sample is particularly well determined.Preferably probesets 540A/540C, and/or 513B/513D and/or 504A/504B,and/or 708K/708L/708M/708N as depicted in FIG. 3C and/or 3D are used todetect KIR2DS3 and/or KIR2DS4 polymorphisms. With probes selected fromFIG. 3 predisposition to leukemia subtypes is thus particularly welldetermined.

Therefore, in one embodiment the invention provides a method fordetermining predisposition to leukemia of an individual comprisingdetermining the presence or absence of KIR2DS4 and/or KIR2DS3 in anucleic acid sample of said individual with at least one probeset listedin FIG. 3A, 3B, 3C and/or 3D, wherein the presence of KIR2DS4 isindicative for a predisposition for chronic myelogenous leukemia and theabsence of KIR2DS3 is indicative for a predisposition for acutelymphoblastic leukemia. In a preferred embodiment probe set 540A/540C,and/or 513B/513D and/or probe set 504A/504B, and/or 708K/708L/708M/708Nas depicted in FIG. 3C and/or 3D are used for determining the presenceor absence of KIR polymorphisms. As used herein, the term “nucleic acidsample” means a sample comprising nucleic acid. Said sample may ofcourse further comprise other components, such as for instance proteins.Preferably, nucleic acid is at least partly isolated from said samplebefore being subjected to a method according to the present invention.

Association between KIR polymorphisms and inflammatory bowel disease(IBD) and/or Crohn's disease have been established as well (Hollenbachet al 2009). The KIR2DL2/KIR2DL3 heterozygous genotype predisposes orprotects from Crohn's disease depending on the presence of their HLA-Cligands. KIR2DL2/KIR2DL3 heterozygosity in combination with C1predisposes to Crohn's disease whereas KIR2DL2/KIR2DL3 heterozygosity incombination with C2 protects from IBD and/or Crohn's disease.KIR2DL2/KIR2DL3 heterozygosity in combination with C1/C2 heterozygosityhas an intermediate effect on predisposition (Hollenbach et al 2009).Non-limiting examples for determining the presence or absence of C1and/or C2 are detecting nucleic acid sequence(s) encoding C1 and/or C2protein using for instance a nucleic acid amplification reaction ordetecting C1 and/or C2 protein using for instance Western blot analysis.

The present invention provides probes that are particularly suitable fordetecting KIR genes, including KIR2DL2 and KIR2DL3. Thus, with probesaccording to the present invention selected from FIG. 3A, 3B, 3C and/or3D KIR2DL2/KIR2DL3 heterozygosity in a sample is particularly welldetermined. Preferably probeset 415B/415C/415D and/or 417A/417B/417Cand/or probeset 420A/420B, and/or 706A/706B as depicted in FIG. 3Cand/or 3D are used to detect KIR2DL3 and/or KIR2DL2 polymorphisms. Withprobes selected from FIG. 3 predisposition to Crohn's disease is thusparticularly well determined.

Therefore, in one embodiment the invention provides a method fordetermining predisposition to IBD and/or Crohn's disease of anindividual comprising determining the presence or absence of KIR2DL2and/or KIR2DL3 in a nucleic acid sample of said individual with at leastone probeset listed in FIG. 3A, 3B, 3C and/or 3D, and determining thepresence of absence of HLA C1 and/or C2 ligand in a sample of saidindividual, wherein KIR2DL2, KIR2DL3 heterozygosity in combination withC1 homozygosity is indicative for a predisposition for Crohn's disease,and KIR2DL2, KIR2DL3 heterozygosity in combination with C2 homozygosityis indicative for protection for Crohn's disease. In a preferredembodiment probe set 415B/415C/415D and/or 417A/417B/417C and/or probeset 420A/420B and/or 706A/706B as depicted in FIG. 3C and/or 3D are usedfor determining the presence or absence of KIR polymorphisms.

Copy number variation of KIR2DL3, KIR3DL1 and KIR3DS1 is correlated tothe course of disease in chronic infection, such as retroviralinfection, herpes virus infection, and hepatitis virus infection, morein particular HIV, CMV, EBV, HSV, HBV and HCV (Martin et al 2007 andKhakoo et al 2004). A higher copy number of KIR3DL1 and/or KIR3DS1 in anindividual is indicative for an improved course of the disease and/orresponse to treatment of chronic infection as compared with a low copynumber of KIR3DL1 and/or KIR3DS1 in an individual and a low copy numberof KIR2DL3 in an individual is indicative for an improved course of thedisease and/or response to treatment of chronic infection as comparedwith a high copy number of KIR2DL3 in an individual. Thus, a higher copynumber of KIR3DL1 and/or KIR3DS1 in an individual is indicative for anincreased survival in chronic infection and a lower copy number ofKIR2DL3 in an individual is indicative for increased survival in chronicinfection.

The present invention provides probes that are particularly wellsuitable for determining copy number variation of KIR genes, includingKIR3DL1 and KIR3DS1. Thus, with probes according to the presentinvention selected from FIG. 3A, 3B, 3C and/or 3D the copy number ofKIR3DL1 and KIR3DS1 and KIR2DL3 in a sample is particularly welldetermined. Preferably probe sets 409A/409B/409C, and/or711A/711B/711C/711D and/or 418A/418B/418D, and/or 709C/709D/709E/709Gand/or probe set 415B/415C/415D and/or 417A/417B/417C as depicted inFIG. 3C and/or 3D are used to estimate the copy number of KIR3DL1 and/orKIR3DS1 and/or KIR2DL3. With probes selected from FIG. 3 susceptibilityof an individual to course of disease and/or response to treatment inchronic infection is thus particularly well determined.

Therefore the invention provides method for determining susceptibilityof an individual to course of disease and/or response to treatment inchronic infection, preferably retroviral infection, herpes virusinfection, and hepatitis virus infection, comprising determining thecopy number of KIR2DL3, KIR3DL1 and/or KIR3DS1 in a nucleic acid sampleof said individual with at least one probeset listed in FIG. 3A or 3B or3C or 3D, wherein a high KIR3DL1 and/or KIR3DS1 copy number in anindividual is indicative for an improved course of disease and/orresponse to treatment of chronic infection as compared with a low copynumber of KIR3DL1 and/or KIR3DS1 in an individual and a low KIR2DL3 copynumber in an individual is indicative for an improved course of diseaseand/or response to treatment of chronic infection as compared with ahigh copy number of KIR2DL3 in an individual. Preferably said chronicinfection comprises HIV, CMV, EBV, HSV, HBV and HCV. In a preferredembodiment probeset 409A/409B/709D/409C, and/or 711A/711B/711C/711Dand/or 418A/418B/418D, and/or 709C/709E/709G and/or probe set415B/415C/415D and/or 417A/417B/417C as depicted in FIG. 3C and/or 3Dare used for determining the copy number of KIR genes.

The presence of KIR2DS4 in a donor is correlated totransplantation-related outcome measures, such as mortality,graft-versus-host, graft-versus-tumor and grafted organ survival inrecipients after transplantation. The presence of KIR2DS4 in a donor isindicative for reduced mortality, reduced graft-versus-host, increasedgraft-versus-tumor and increased grafted organ survival in recipientsafter transplantation as compared to the absence of KIR2DS4 in a donor.The present invention provides probes that are particularly wellsuitable for determining copy number variation of KIR genes, includingKIR3DL1 and KIR3DS1. Thus, with probes according to the presentinvention selected from FIG. 3A, 3B, 3C and/or 3D the copy number ofKIR2DS4 in a sample is particularly well determined. Preferably probesets 504A/504B, and/or 708K/708L/708M/708N as depicted in FIG. 3C and/or3D are used to the presence or absence of KIR2DS4. With probes selectedfrom FIG. 3 predisposition to transplantation-related outcome measuresis thus particularly well determined

Therefore the invention provides a method for determining predispositionto transplantation-related outcome measures, such as mortality,graft-versus-host, graft-versus-tumor and grafted organ survival of arecipient after transplantation, comprising determining the presence orabsence of KIR2DS4 in a nucleic acid sample of a donor for saidrecipient with at least one probeset listed in FIG. 3A or 3B or 3C or3D, wherein the presence of KIR2DS4 in said donor is indicative for areduced mortality, a reduced graft-versus-host reaction, an increasedgraft-versus-tumor reaction and an increased grafted organ survival insaid recipient as compared to the mortality, graft-versus-host reaction,graft-versus-tumor reaction and grafted organ survival of a recipientwith a donor wherein KIR2DS4 is absent. In a preferred embodimentprobeset 504A/504B, and/or 708K/708L/708M/708N as depicted in FIG. 3Cand/or 3D are used for determining the presence or absence of KIRpolymorphisms.

A correlation has been established between the copy number of KIR2DL2and KIR2DS2 and rheumatoid arthritis (RA) with extra-articularmanifestations and rheumatoid vasculitis. A higher copy number ofKIR2DL2 and/or KIR2DS2 in an individual was demonstrated to bepredisposing for rheumatoid arthritis with extra-articularmanifestations and rheumatoid vasculitis (Majorczyk et al 2007, Yen etal 2001). Additionally, rheumatoid arthritis patients positive forKIR2DL3 and negative for KIR2DS3 had earlier disease diagnosis(Majorczyk et al 2007).

The present invention provides probes that are particularly wellsuitable for determining the presence or absence and copy numbervariation of KIR genes, including KIR2DL2, KIR2DS2, KIR2DL3 and KIR2DS3.Thus, with probes according to the present invention selected from FIG.3A, 3B, 3C and/or 3D the presence or absence and copy number of KIR2DL2,KIR2DS2, KIR2DL3 and KIR2DS3 in a sample is particularly welldetermined. Preferably probe sets 420A/420B, and/or 706A/706B and/orprobe set 703A/703B/703C, and/or 544A/544B as depicted in FIG. 3C and/or3D are used to estimate the copy number of KIR2DL2 and/or KIR2DS2.Preferably probe sets 415B/415C/415D and/or 417A/417B/417C and/or probeset 513B/513D and/or 540A/540C as depicted in FIG. 3C and/or 3D are usedto estimate the copy number of KIR2DL3 and/or KIR2DS3. With probesselected from FIG. 3 susceptibility of an individual to rheumatoidarthritis (RA) with extra-articular manifestations and rheumatoidvasculitis is thus particularly well determined.

Therefore in one embodiment the invention provides a method fordetermining predisposition to rheumatoid arthritis with extra-articularmanifestations and rheumatoid vasculitis of an individual comprisingdetermining the copy number of KIR2DS2 and/or KIR2DL2 in a nucleic acidsample of said individual with at least one probeset listed in FIG. 3A,3B, 3C and/or 3D, wherein a high copy number of KIR2DS2 and/or KIRDL2 insaid individual is indicative for a predisposition for rheumatoidarthritis with extra-articular manifestations and rheumatoid vasculitisas compared with a low copy number of KIR2DL2 and/or KIR2DS2 in anindividual. In a preferred embodiment probeset 420A/420B, and/or706A/706B and/or probe set 703A/703B/703C, and/or 544A/544B as depictedin FIG. 3C and/or 3D are used for determining the copy number of KIRgenes.

Finally, a correlation has been found between the presence or absence orcopy number of KIR genes and predisposition to autoinflammation, such asHLA-B27-related enthesitis-related arthropathy and reactive arthritis,psoriasis, in individuals. For instance, KIR3DL2 is increased inspondylarthritides and juvenile enthesitis-related arthritis (Chan et al2005, Brown 2009). The present invention provides probes that areparticularly well suitable for determining the presence or absence andcopy number variation of KIR genes. Thus with probes selected from FIG.3 susceptibility of an individual to autoinflammation, such asHLA-B27-related enthesitis-related arthropathy and reactive arthritis,psoriasis is particularly well determined.

Therefore, in one embodiment the invention provides a method fordetermining predisposition to autoinflammation, preferablyHLA-B27-related enthesitis-related arthropathy and reactive arthritis,psoriasis, in individuals comprising a) determining the presence orabsence and/or copy number of a KIR gene indicative for said disorder ina nucleic acid sample of said individual with at least one probesetlisted in FIG. 3A or 3B or 3C or 3D, and b) correlating the resultobtained in step a) with presence or absence of said predisposition.

In another embodiment the invention provides a method for determiningpredisposition to spondylarthritides and/or juvenile enthesitis-relatedarthritis of an individual comprising determining the copy number ofKIR3DL2 in a nucleic acid sample of said individual with at least oneprobeset listed in FIG. 3A, 3B, 3C and/or 3D, wherein a high copy numberof KIR3DL2 in said individual is indicative for a predisposition forspondylarthritides and/or juvenile enthesitis-related arthritis ascompared with a low copy number of KIR3DL2 in an individual. In apreferred embodiment probeset 404A/404B, and/or 538A/538B/538D asdepicted in FIG. 3C and/or 3D are used for determining the copy numberof KIR genes.

The invention is further explained in the following examples. Theseexamples do not limit the scope of the invention, but merely serve toclarify the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. A) Schematic outline of a conventional MLPA reaction. The figureis adapted from www.mpla.com.

FIG. 1B) illustrates the use of two ligation sites in one probe set, todetect two SNP's at the same time with one probe set (a Tri-Lig probe)on a specific target sequence. If the correct SNP's are present at bothligation sites, the three probe parts will become ligated together toresult in one PCR product, as shown at the bottom left. If an incorrectSNP is present on one or both ligation sites, no PCR product will beformed, as shown at the bottom right.

FIG. 1C) illustrates the use of two ligation sites in one Tri-Lig probe,to detect one particular gene, KIR3DL1*024N, in the background of allother KIR3DL1 WT alleles at the first ligation site, and all other KIRgenes at the second ligation site. The 1a probe detects all WT KIR3DL1alleles (1a) whereas the 1b probe only detects the KIR3DL1*024N allele(1b), due to a different SNP at the first ligation site. The partial KIRgene sequences 2 to 12 are not detected by the 1a and 1b probes, becausethese probes are only specific for KIR3DL1 genes at the second ligationsite due to a different SNP at the second ligation site.

FIG. 2. IUB nucleotide codes of degenerate bases

FIG. 3 KIR-specific probe sets. A) KIR probe mix 1. Bold nucleotidesrepresent probes that are part of a probe set consisting of three probesused for detection of two SNPs, B) KIR probe mix 2. Bold nucleotidesrepresent probes that are part of a probeset consisting of three probesused for detection of two SNPs, C) extended KIR probe mix 1. Boldnucleotides represent primer binding sites. KIR genes in which two SNPsare detected using one probe set according to the invention, consistingof three probes are depicted in FIG. 13, D) extended KIR probe mix 2.Bold nucleotides represent primer binding sites. KIR genes in which twoSNPs are detected using one probe set of this probe mix, consisting ofthree probes are depicted in FIG. 13, E) control probe mix.

FIG. 4. The KIR protein structures. Depicted as large ovals are theextracellular Ig-like domains, as squares the ITIMs and as small lightgrey circles the charged residues on the cytoplasmic tail (IPDKIRdatabase). Inhibitory KIRs and activating KIRs are indicated by a “+”and “−”, respectively.

FIG. 5. Exon structure of KIR3DL1. Exons are depicted with black boxesand introns with lines and are draw approximately to scale (Vilches etal, 2002).

FIG. 6. The organization of KIR locus. a: Framework genes KIR3DL3,KIR2DL4 and KIR3DL2 are in black and are found at the beginning, nearthe middle and at the end of the locus. The pseudogenes KIR2DP1 andKIR3DP1 (which is also a framework gene) in white and black,respectively, and the regions between the framework genes are variableand these KIR genes are in grey, with activating KIRs with black lettersand inhibitory KIRs in white. b: One example of haplotype A. c: Anexample of haplotype B (Parham et al, 2003).

FIG. 7. The pedigrees of 12 families from the KIR reference panel I (thefamilies 1347 and 1349 are depicted in FIGS. 11 and 12, respectively).The four numbers on top of the pedigree is the CEPH family number andthe numbers in the shapes is the individual number, these numberscorrespond with the numbers in table 4. The letters below the shapeindicates the haplotypes and can be found in the legend next to thepedigree.

FIG. 8. Electropherogram of probe set 1. The peak patterns of the probeson two donors: 8080 (top) and 5911 (bottom). All 17 KIR probe peaks arepresent on donor 8080 and 10 KIR probe peaks on donor 5911. In alldonors the nine control probes (Ctr2-10) and the probes on the fourframework genes: KIR3DL3, KIR3DP1, KIR3DL2, and KIR2DL4 (indicated withthe black arrows) generated a signal. Electropherogram of probes set 2were similar for these two probe groups (data not shown).

FIG. 9. Comparison of peak intensities of the probe 2DS2 (black arrows)between a true positive for KIR2DS2 (top) and a false positive (bottom).

FIG. 10. The peak profiles of the probes 2DL5 (left arrows) and 2DL5A(right arrows). Top: a sample which is positive for KIR2DL5 indicated bythe presence of the peak from probe 2DL5 and the peak from 2DL5A cannotbe distinguished in the presence of KIR2DL5A or 3DP1*004. Bottom: thissample is negative for KIR2DL5 indicated by the absence of the probe2DL5 and the peak of 2DL5A indicates the presence of KIR3DP1*004.

FIG. 11. The pedigree of family 1347.

A) Left: The numbers of the individuals in top left pedigree correspondwith the numbers of the DNA samples in the table. At the bottom thehaplotype is denoted in letters and the legend for the haplotype isdisplayed below (www.ihwg.org). The CNV of some of the genes wherequantified different by each of the two probe sets, the number before‘/’ is for probe set 1 and after for probe set 2.

B1) Interpretation based on SSP-PCR data from CEPH-IHWG and theconventional KIR haplotype model (see alsohttp://www.ncbi.nlm.nih.gov/projects/gv/mhc/xslcgi.fcgi?id=1347&cmd=kirped&locus_group=1).

B2) Novel haplotype model based on SSP-PCR data obtained from CEPH-IHWG(http://www.ncbi.nlm.nih.gov/projects/gv/mhc/xslcgi.fcgi?id=1347&cmd=kirped&locus_group=1).

B3) Copy number variation of KIR genes, determined using SSP-PCR dataobtained from CEPH-IHWG based on the conventional KIR haplotype model(table 1) and the novel KIR haplotype model (table 2) and copy numbervariation of KIR genes, determined by KIR-MLPA using the extended probesets 1 and 2 and the novel KIR haplotype model (table 3).

FIG. 12. The pedigree of family 1349.

A) Left: The numbers of the individuals in top left pedigree correspondwith the numbers of the DNA samples in the table. At the bottom thehaplotype is denoted in letters and the legend for the haplotype isdisplayed below (www.ihwg.org). The CNV of some of the genes wherequantified different by each of the two probe sets, the number before‘/’ is for probe set 1 and after for probe set 2.

B1) Interpretation based on SSP-PCR data from CEPH-IHWG and theconventional KIR haplotype model (see alsohttp://www.ncbi.nlm.nih.gov/projects/gv/mhc/xslcgi.fcgi?id=1347&cmd=kirped&locus_group=1).

B2) Novel haplotype model based on SSP-PCR data obtained from CEPH-IHWG(http://www.ncbi.nlm.nih.gov/projects/gv/mhc/xslcgi.fcgi?id=1347&cmd=kirped&locus_group=1).

B3) Copy number variation of KIR genes, determined using SSP-PCR dataobtained from CEPH-IHWG based on the conventional KIR haplotype model(table 1) and the novel KIR haplotype model (table 2) and copy numbervariation of KIR genes, determined by KIR-MLPA using the extended probesets 1 and 2 and the novel KIR haplotype model (table 3).

FIG. 13. Detection of KIR alleles and KIR copy number variation.

EXAMPLES Example 1

This Example presents a new method for KIR genotyping.

KIRs are expressed by natural killer (NK) cells and a subset of T cells.NK cells are cells of the lymphoid lineage, but display noantigen-specific receptors. Their main function is to monitor host cellsfor the presence of MHC class I molecules and this is important for e.g.distinguishing healthy cells from virus-infected or tumors cells. A lowexpression of MHC class I molecules on host cells, which may forinstance occur during viral infections as a result of virus-mediateddown regulation to prevent presentation of viral peptides to CD8 Tcells, stimulate NK cells to launch cytotoxic attack. This phenomenon isalso known as the “missing self” theory.

NK cells express a variety of receptors that mediate interactions withMHC class I molecules, including members of the KIRs and CD94/NKGreceptor multigene families. Interaction between MHC class I moleculesand these receptors regulates NK cytotoxicity generally through thegeneration of inhibitory signals. The composition between KIR andCD94/NKG families of humans and mice differs considerably, with KIRsconstituting the most in genetic and gene number variation in man.

KIRs were first discovered in their role in fighting virus infections bynatural killer cells, but they are also expressed by a subset of Tcells. The KIR gene cluster is located at chromosome 19q13.4 within theleukocyte receptor complex (LCR) and spans a region of about 150 kb. Upto 15 genes plus two pseudogenes have been identified to date.Characteristic of the KIR gene cluster is the variable gene content andan extensive degree of allelic gene variants. The gene content betweenunrelated individuals can differ considerably in the amount of KIR(pseudo)genes present, but also in the numbers of activating andinhibitory (pseudo)genes. Contractions and expansions by non-reciprocalrecombination are the major mechanism behind KIR diversification. KIRscan be divided into two haplotypes, A and B in which haplotype B has agreater variety in gene content and contains more activating KIR genes.Studies of different ethnic populations show significant differences inthe distribution of these two haplotypes. The selective pressures, suchas exposure to different pathogens and rapidly evolving MHC class Imolecules appear to be the forces behind such a gene diversification. Afunctional analog is the Ly49 gene family in mice, but KIRs and Ly49 arestructurally distinct proteins. KIRs have been identified in differentprimate species, but they are species-specific and differ in genecontent among various species. These findings provide evidence for arapid evolution and expansion of this gene family.

Another level of relevant variation is the level of expression of KIRsby individual NK cells. Each NK cell expresses only a subset of its KIRgene repertoire and the presence of HLA ligands seems to influence thefrequency of NK cells expressing the cognate ligand. A higher frequencyof NK cells expressing inhibitory KIRs in individuals have been found,when their cognate HLA ligand is present. The ligands of some KIRs, inparticular those with activating potential remain to be determined.

Some of these activating KIRs seem to have lower affinity for theircognate HLA class I ligands in comparison with their related inhibitoryreceptors.

KIRs have been associated with several diseases, but due to the geneticdiversity between and in populations and the differences in KIRexpression by NK cells, a clear understanding of their role has yet tobe defined. KIRs have been reported to play a role in allogeneichematopoietic stem cell transplantation (HSCT), which is used in thetreatment of leukemia. It was suggested that an intentional mismatchbetween donor KIR and recipient HLA ligands would allow for a graftanti-tumor effect. KIR3DS1 and KIR3DL1 have been reported to beassociated with slower progression to AIDS and several other virusinfections, such as Hepatitis C virus (HCV), human cytomegalovirus(CMV). Also the protozoan infection with Plasmodium falciparumimplicated roles for KIRs in malaria. In autoimmune and inflammatoryconditions, certain KIRs and cognate ligand potentially results inhigher susceptibility or protection of the host.

The KIR Gene Cluster

The KIR acronym originally stood for killer cell-inhibitory receptor,because the first KIR discovered had an inhibiting effect on NK cells.To date, KIR is an abbreviation for Killer-cell Immunoglobulin-likeReceptor, as this family includes both inhibitory and activatingreceptors. The HUGO Genome Nomenclature Committee (HGNC) is responsiblefor the naming of KIR genes. Currently KIR gene family consists of 15genes and 2 pseudogenes, listed in Table 1 (Marsh et al, 2002). KIRgenes are named after the protein structure they encode. The “D” denotes“Domain” and the number 2 or 3 before it indicates the number ofextracellular Ig-like domains. “L” indicates a “Long” cytoplasmic tailand “S” indicates a “Short” cytoplasmic tail and the “P” indicates a“pseudogene”. The number behind the letter L or S denotes the geneencoding for this structure. Thus KIR2DL1 encodes for a structure withtwo Ig-like domains and a long cytoplasmic tail. KIR2DL5A and KIR2DL5Bare exceptions; they were initially identified as one gene KIR2DL5.However these two structurally similar variants are discovered to belocated on different regions of the KIR gene cluster and can beinherited separately (Gomez-Lozano et al, 2002).

The KIRs that possess long cytoplasmic tails transduce inhibitorysignals to the NK cell, owing to the two immunoreceptor tyrosine-basedinhibitory motifs (ITIMs) (FIG. 4). Binding of these receptors with HLAclass I molecules leads to phosphorylation of the tyrosine residueswithin the ITIM. Tyrosine phosphatase (SHP-1) is then recruited andactivated by the ITIM and prevents or inhibits phosphorylation eventswhich are associated with cellular activation. NK-cell mediatedcytotoxicity and cytokine secretion inhibition are the main downstreameffects. Short cytoplasmic tails lack the ITIM and possess a basiccharged amino acid, such as lysine in the transmembrane domain. Thispositively charged amino acid residue allows association with an adaptormolecule, such as DAP12. DAP12 has one immunoreceptor tyrosine-basedactivation motif (ITAM). When the tyrosine residues in the ITAM arephosphorylated a docking site for SH2 domain of ZAP70 and Syk tyrosinekinase is generated. The action of these kinases triggers a downstreamtransduction cascade that promotes NK-mediated cytolysis (Middleton etal, 2005). KIR2DL4 is unique among KIRs, as it possesses a longcytoplasmic tail with a charged amino acid arginine in the transmembraneregion. KIR2DL4 might therefore be capable of eliciting both activatingas well as inhibitory signals.

Exon and Intron Structure

The KIR3DL1 and KIR3DL2, with three extracellular Ig-like domainsrepresent the prototypical KIR from which all the others can be derived.KIR genes are organized in nine exons, the order of these exonscorresponding to the different functional regions of the protein (FIG.5). The first two exons encode the signal peptide, exons 3, 4 and 5encode the Ig-like domain, D0, D1 and D2, respectively. Exon 6 encodesthe stem or linker that connects the D2 domain with the transmembraneregion that is encoded by exon 7. Exons 8 and 9 encode the cytoplasmictail. Type 1 KIRs have two Ig-like domains D1 and D2, KIR2DL1-3 andKIR2DS1-5. The protein products of type 1 lack the D0 domain becauseexon 3 is a pseudo-exon. This exon is spliced out of the RNA transcript,possibly due to a three-base-pair deletion. Type 2 KIRs have the D0 andD2 domains, KIR2DL4-5, exon 4 is absent in these KIR genes, resulting ina protein without D1 domain.

In KIR2DP1 exon 3 is a pseudoexon and exon 4 has an early stop codon. IfKIR2DP1 would be transcribed this could result in a KIR protein withonly a single Ig (D2) domain. In KIR3DP1 exon 2 is missing due to adeletion. The exons encoding for the stalk, TM and cytoplasmic regionsare also absent. The three exons coding for the Ig-like domains areintact, however the leader sequence is missing. No transcripts have beenfound for KIR2DP1 (Trowsdale et al, 2001) and KIR3DP1, the latest one isnormally silent, but a recombination of KIR2DL5A and KIR3DP1 have beenfound to be transcribed and is predicted to be secreted rather thananchored to the cell membrane (Gómez-Lozano, 2005).

Genotypes

Uhrberg et al. (Uhrberg et al, 1997) identified that the KIR locus inhumans appeared to be polygenic and polymorphic. Individuals have avariable KIR gene content, achieved through differences in number oftotal KIR genes and differences in the amount of activating andinhibitory KIR genes. The mechanism behind the KIR diversification isnon-reciprocal recombinations between non-allelic genes leading toexpansion and contractions of the KIR locus. Also reciprocal crossingover events are postulated to contribute to the diversity. The KIR locuscan be separated into two parts with KIR3DL3 on the centromeric end andthe central KIR3DP1 on one half, and KIR2DL4 in the central and KIR3DL2on the telomeric end on the other half. Inside these two parts of KIRlocus, genes are located that are in much stronger linkagedisequilibrium, supporting a homologous recombination event (Uhrberg2005).

Studies worldwide using genomic DNA to determine the presence or absenceof KIR genes in populations have contributed to an extensive amount ofKIR-genotype profiling data. These studies show a difference infrequency of KIR genes in populations of different ethnic backgroundsand can be found on www.allelefrequencies.net. The methods used for KIRgenotyping are polymerase chain reaction with sequence-specific primers(PCR-SSP), sequence-specific oligonucleotide probes, PCR (PCR-SSOP),multiplex PCR, automated sequencing and mass spectrometry.

Haplotypes

KIR genes can be divided in the haplotypes A and B (Carrington et al,2003). Both haplotypes contain the framework genes KIR3DL3, KIR3DP1,KIR2DL4 and KIR3DL2. These genes are conserved and are virtually presentin every individual. Haplotype A is uniform in terms of gene content andis composed of five inhibitory genes (KIR3DL3, KIR2DL3, KIR2DL1,KIR2DL4KIR3DL1 and KIR3DL2, and only one activating KIR2DS4, as shown inFIG. 6. However the central framework gene KIR2DL4 may have anactivating function. On the other hand, there are haplotypes A thatpossess null variants of both KIR2DS4 and KIR2DL4 that are not expressedon the cell surface and technically these haplotypes contain virtuallyno functional activating KIR.

Haplotype B is more variable than haplotype A and is characterized byone or more of the following genes: KIR2DS2, KIR2DL2, KIR2DL5, KIR2DS3,KIR3DS1, KIR2DL5A, KIR2DS5 and KIR2DS1, conversely haplotype A ischaracterized by the absence of these genes. The frequency of bothhaplotypes is relatively even among populations of different ethnicbackground. It is possible that some haplotypes cannot be placed inthese two categories, as the definition of haplotypes varies betweenauthors and hybrids of haplotypes are possible (Vilches et al, 2002).Distinction between A and B haplotypes is useful in biological andmedical settings, as haplotype B have more genes that encode foractivating KIR than haplotype A. The haplotypes have been constructed byfamily segregation analysis, genomic sequencing and gene-order analysis(Shilling et al, 2002). FIG. 6 depicts the organization of a KIR locus.

Gene Variation

Adding another level of genetic diversity to the KIR family is theextensive degree of gene variations, which are exhibited by all KIRgenes. Allelic diversity is generated by substitutions of nucleotides,recombination or gene conversion and point mutations. Activating KIRsand inhibitory KIRs share a high sequence homology. Activating KIRs arebelieved to be derived from inhibitory KIRs by alterations in sequence,creating a charged residue upstream of a stop codon and an eliminationof ITIMs. Due to their younger evolution, allelic diversity ofactivating KIRs is quite limited when compared to inhibitory KIRs, butthe variation of activating receptors across ethnic populations is moreextensive.

Currently a total of 335 KIR alleles have been identified and can befound at the website: http://www.ebi.ac.uk/ipd/kir (table 2). KIR allelesequences are denoted by an asterisk after the gene name. Differences inthe encoded protein sequences are distinguished by the first threedigits, the next two digits are used to denote alleles that differ bysynonymous differences within the coding sequence (i.e. not resulting inamino acid substitutions) and the last two digits are used for allelesthat have differences in the noncoding region, such as introns andpromoters. Thus, 3DL1*009 and 3DL1*010 are alleles that encode differentprotein products and 3DL1*00101 and 3DL1*00102 are alleles that encodethe same protein product, but these alleles differ by a synonymous DNAsubstitution within the coding region (Marsh et al, 2002).

Expression and HLA

The ligands for inhibitory KIRs are MHC class I molecules, which areconstitutively expressed by most healthy cells, but can bedown-regulated in tumors and infected cells allowing killing by NKcells. Interaction of MHC with inhibitory receptors ensures tolerance ofNK cells towards self. MHC class I molecules are encoded by humanleukocyte antigen (HLA) genes that are located at chromosome 6p21.3 andare polymorphic and display significant variations. KIR genes and HLAgenes segregate independently during meiosis, because they are locatedon different chromosomes. This can lead to interesting HLA and KIRcombinations inherited by one individual, but to obtain a functionalinteraction between receptor and the cognate ligand, they need to beexpressed together. This raises the question whether a correlationexists between the genes encoding KIR and HLA. The ligand specificityfor activating KIRs is not well defined. The ligands of some activatingKIRs have not been identified yet. The activating receptors of KIR2DS2and KIR2DS1 were reported to have a lower affinity of binding to HLA-Cthan those of their closely related inhibitory receptors. It is alsopossible that non-HLA ligands exist for these activating KIRs. The KIRswith a defined cognate ligand are presented in table 3.

The KIR surface protein repertoire in an individual is mainly determinedby the KIR genes. Hence, a lack of expression is more likely caused bythe lack of that gene than by a down-regulation. KIR genes are expressedby NK cells in a clonal manner, each individual NK cell within a personpossesses a different combination of KIRs, with a subset of the totalKIR gene repertoire being expressed on each individual. KIR2DL4 is onenotable exception; this gene is ubiquitously expressed on NK cells. Thefrequency of each expressed KIR may differ between individuals, but isstable over time. For example the gene KIR2DL1 may be expressed on 50%of the NK cell population of individual A, while in individual B theexpression of KIR2DL1 is found to be 14% of its NK cell population. Oneexplanation for this difference could be that particular alleles of agene are expressed more frequently due to the presence of multiplecopies of a gene.

This Example presents a new method for KIR genotyping with multiplexligation dependent probe amplification (MLPA). With this method a rapidand convenient way of KIR genotyping is performed and also the relativenumber of copies of the KIR genes is quantified. Copy number variation(CNV) accounts for a substantial amount of genetic variation, resultingin significant phenotypic variations in e.g. transcript levels andtherefore are of functional relevance.

We developed two synthetic MLPA probe sets for the typing of 16 out ofthe 17 KIR genes KIR2DL1-5, KIR2DS1-5, KIR3DL1-3, KIR3DS1, KIR3DP1 andKIR2DP1. The probes for the KIR genes were designed for different locito detect most of the alleles. Probesets 1 and 2 are listed in FIGS. 3Aand 3B. The specificity of the probes was validated by comparison of thesamples for the KIR genotypes obtained with PCR-SSOP and PCR-SSPmethods, and the ability of the probes to quantify relative gene copynumbers was examined with 12 families, each consisting of two parentsand two offspring, which have been genotyped for most KIR alleles.

Materials & Methods DNA Selection/Isolation

DNA from unrelated randomly selected Caucasian donors was obtained forthis study to test the peak profile of the probes. For the validation ofthe probes five SSP-PCR KIR typed genomic DNA samples and 11 EBVtransformed B cell lines from the 10^(th) InternationalHistocompatibility Workshop were used (Cook et al, 2003), JVM, T7507,OLGA, SAVC, JBUSH, BM16, LBUF, AMALA, BM90, TAB089 and KAS116. The KIRReference Panel I from the IHWG containing 48 samples from 12 Centrede'Etude du Polymorphism Humain (CEPH) families—including 2 parents and2 children (table 4: KIR typing of the 48 samples and FIG. 7: thepedigrees)—also served this purpose, but its main purpose was todetermine the ability of copy number quantification of the probes.Genomic DNA and the DNA from the Cell lines were isolated with Qiagen(blood kit) according to the manufacturer's instructions.

Probe Design

Probes were designed according to general instructions(www.mlpa.com/protocols.htm). All the probes were manufactured byInvitrogen (Carsblad, Calif.). The sizes of the probes after ligation(“ligated probes”) are spaced four to five nucleotides apart, toseparate each amplification product on the sequence type gels,amplification product size ranged from 95 to 223 nucleotides. All MLPAprobes contain a PCR primer sequence, which is recognized by a universalprimer pair. PCR primer sequences were: forward 5′-GGGTTCCCTAAGGGTTGG-3′and reverse 5′-TCTAGATTGGATCTTGCTGGCAC-3′.

The KIR probes were designed to identify and discriminate between the 17KIR genes listed in table 1, with exception of KIR2DL5B. No specificprobe could be designed for this gene. The probe for KIR2DL5 now,detects both KIR2DL5A and KIR2DL5B genes. In addition probes onalternative sequences and intron sequences were designed, using basiclocal alignment sequence tool searches and the IPD/KIR Database,http://www.ebi.ac.uk/ipd/kir. The sizes of the KIR probes can be foundin tables 5 and 6.

The targets of the nine control probes are on conserved genes in thehuman genome, FGF3, BCAS4, LMNA, PARK2, MSH6, GALT, SPG4, IL-4 and NF2.These target genes were tested to show no considerable variation betweendonors in a previous MLPA study at Sanquin. Control 1 and 10 wereinitially 88 bp and 130 bp respectively, but have been elongated to 180bp and 223 bp to distribute the control probes more evenly among the KIRprobes. Table 7 shows the list of the genes and the sizes of the controlprobes.

Competitor probes are designed where the signal of the probe wasoff-scale to be detected by the capillary electrophoresis apparatus andare listed in table 8.

MLPA Reaction

All DNA samples were diluted to 20 ng/μl with water and 5 μl wasdenatured at 98° C. for 5 minutes in 200 μl tubes in a Biometra T-1Thermoblock with heated lid.

MLPA reagents (EK kit 5) were obtained from MRC-Holland (Amsterdam, TheNetherlands). SALSA MLPA buffer (2 μl) and 1-10 fmol of each MLPA probein a probe mixture (1 μl) were added and incubated for 1 minute 95° C.,followed by 16 hours at 60° C. in a total volume of 10 μl. Ligation ofthe hybridized probes was performed by reducing the temperature to 54°C., before adding 32 μl Ligase-65 mix (3 μl ligase buffer A, ligasebuffer B, 1 μl Ligase-65 and 25 μl water) and incubated for 15 min.After inactivating the enzyme at 98° C. for 5 min, 10 μl of the ligasemix was diluted with 4 μl PCR Buffer and 26 μl water at 4° C. in 200 μltubes. For the PCR reaction, 10 μl of polymerase mix (0.5 μl polymerase,2 μl SALSA enzyme dilution buffer, 2 μl SALSA PCR-primers and 5.5 μlwater) was added at 60° C. PCR amplification of the ligated MLPA probeswas performed for 36 cycles (30 sec 95° C., 30 sec 60° C., 60 sec 72°C.) followed by an incubation for 20 min at 72° C.

Electrophoresis

1 μl PCR product is added in new tubes containing 0.4 μl Promega Roxsize standard 60-400 bp+8.6 μl High Definition buffer. The products areseparated by Applied Biostystems Genetic Analyzer 3130XL capillaryelectrophoresis according to its molecular weight and the resultingelectropherogram show specific peaks that correspond to each probe.

Analysis

Data were visualized with Genemapper v3.6 and normalized with Softgenetics Genemarker v1.6, using internal control probe normalization(http://www.softgenetics.com/papers/MLPA). Finally these data wasexported to an Excel file.

Results Detection of Probe Signal

All the MLPA probes were initially tested on randomly chosen donors. Wefirst examined if the probes would generate a signal and if thesesignals corresponded with the expected size of each probe. The controlprobe peaks and the probe peaks for the four framework genes, KIR2DL4,KIR2DL3, KIR3DL3 and KIR3DP1, occurred in all samples, as expected. KIRgene content variation between individuals was observed when differentsamples were compared, FIG. 8. The probe intensity is denoted byarbitrary units (AU) on the y-axis and the probe size is expressed onthe x-axis in basepairs (bp). We used the peak height to quantify thedata, while others may suggest probe area.

Secondly, the intensity of the probe signal was examined. The peakpatterns were visualized with Genemapper, to observe the peakintensities before normalization. Genemarker is used to normalize thedata and correct this for the decay of larger probes, but does notindicate where signals are off-scale. It is preferred to have a probesignal between 500-6000 AU in order to obtain a more reliable DQ value.Moreover fluorescent peaks with a signal less than 500 AU may not alwaysbe detected when more probes are added to the reaction. Fluorescentpeaks above 6000 AU can be off-scale to be detected by the sequencer anddecrease the signal of other probes relatively. Several suggestions aredescribed to enhance or lower probe intensity, the nucleotidecomposition next to the PCR primer tag sites and/or the GC content of aprobe are a few factors that can be of influence(www.mlpa.com/protocols.htm). In general competitors are used forreduction of probe signals and a higher probe concentration for anincrease in signal. Competitors are oligonucleotides that are identicalto a part of the MLPA probe without the forward or reverse primersequence, depending whether the left or right part is chosen.

Competitors compete with the MLPA probe for the same target, however noamplification of these ligated probes will occur, since they lack aprimer sequence. The result is that less probe amplification productwill be detected and lower peak intensity is obtained.

Competitors were designed for control probes 2, 3, 4, 7 and 9 and in thefirst place also for the KIR probes 2DL4, 3DL3 (probe set 1) and 3DL2(probe set 2) These probes had a length of 96 bp, 100 by and 108 bp,respectively. However we observed a decrease in peak intensity, more orless corresponding with an increase in probe size. Longer syntheticprobes are more likely to contain a higher proportion of incompleteoligonucleotides. Therefore it seemed to be an option to elongate thelength of probes with high peak intensities and to shorten this forprobes with low peak intensities. Probe 2DL4 was redesigned to 170 bpand 3DL3 to 154 bp and lower peak intensities were the result. The peakgenerated by probe 3DL3 (100 bp) was not affected by its competitor andwas apparently a product of the probe 2DS3 (108 bp), because when thisprobe was removed from the probe set 1, the off-scale signal reduced tonormal. Furthermore competitors with a length of 30 bp had less effectthan those with a length of 50 bp, in which case a higher dosage wasneeded to reduce the probe signal (data not shown).

For probes that failed to generate a signal or for which the signal wasinsufficient, the followings have been performed; a three- to ten-foldconcentration of these probes was used and probes that have a highoverlap in sequence were not included in one probe set. Placing twocytosine nucleotides after the forward primer should increase the probessignal and a tyrosine base should decrease this, reported in the MLPAdesign protocol. However in our experiment, several probes wereredesigned to contain two cytosines after the forward primer and thisdid not produce the same results. Probes that still failed to generate asignal after the aforementioned proceedings and testing on lager numberof donors were replaced by probes on the reverse strand of the targetgene or by probes that have a different target location on that gene.

The frequencies of each KIR gene probe peak on the tested samples werecompared with the KIR gene frequencies in Caucasian population availableon www.allelefrequencies.net (table 9). Probes with observed frequenciesthat were contradicted by the population frequencies were assumed togive false negative or false positive results and were replaced by newdesigns. These were assumed to be caused by gene variation at theligation sites of the probe.

The list of the alleles that can be detected by the KIR probes and thecoverage of the total KIR alleles by the probes are shown in table 10.

Other Factors Interfering with Peak Intensities

Probe Quality

We experienced differences probe quality by probes that weremanufactured at different companies. The nine control probes wereinitially ordered from Biolegio (www.biolegio.com) which had alsosupplied these for the C4 MLPA project previously done here. All the KIRMLPA probes were ordered at Invitrogen (www.invitrogen.com). The controlprobe set was separated in two mixes, control probes 1 (IL-4), 2 (FGF3),3 (BCAS4), 4 (LMNA), 5 (PARK2) and 7 (MSH6) in one and the controlprobes 8 (GALT), 9 (SPG4) and Ctrl 10 (NF2) in the other. Theconcentration needed for each control probe varied and ranged from 0.5fmol to 6 fmol and also different concentrations of competitors wereneeded.

The control probes used for the KIR MLPA were ordered from Invitrogen.Only 1 fmol is needed for each control, with the exception of controlprobe 5 (3 fmol) in order to obtain the same peak intensity as mentionabove and the probes do not need to be separated into two mixes. Due tothe better probe quality, time is saved in producing the probe sets.

Template DNA Amount

A MLPA reaction with 50 ng of DNA was performed and compared with 100 ngthat is used throughout this study. MLPA reactions using a DNA amount of20 ng have been reported by Schouten et al. (Schouten et al, 2002). Whenthe peak profiles were compared, no striking differences between thesetwo reactions were observed. The DQ of the nine control probes werecalculated for each sample and a sample with 100 ng DNA was taken asreference. Seven out of eight samples containing 50 ng of DNA showed aDQ value outside [0.8-1.2] for more than three control probes, rangingfrom [0.3-1.5] within one sample. While all the eight samples of 100 ngDNA had DQ within the acceptable range [0.8-1.2] for all the ninecontrol probes, with exception of one sample that had two control probeDQ value outside this range. Here we conclude that MLPA reactions withdifferent amounts of DNA cannot be compared with each other, because theDQ values of the same sample did not yield the same score with thedifferent DNA amounts.

Next the samples of 50 ng of DNA were compared among, by taking a sampleof 50 ng DNA as reference. The observation was that three of the eightsamples had more than three control probes with a DQ value out of therange of [0.8-1.2]. When the nine control DQ values of one sample wereanalyzed, values between [0.5-1.7] were found. Therefore MLPA reactionscarried out with 50 ng of DNA were considered to be unreliable, as theDQ values of the probes showed a great variation between the samples andwithin one sample, which was not observed with the samples thatcontained 100 ng of DNA. The requirement of higher amounts of DNA forthis study could be explained by the fact that we are using a completelysynthetic probe set in contrast with the probe sets used by Schouten etal (Schouten et al, 2002). Moreover most studies that were carried outwith little amount of DNA often only analyzed chromosomal abnormalities,such as recombination or mutations and did not quantify copy numbers.

Reproducibility

Samples of different runs were not always comparable, when the DQ of thecontrol probes were calculated. The explanation is that the experimentalconditions may vary with each run, due to human acting or differences inprobe signal reproducibility. Therefore, samples within the same run arepreferably normalized and analyzed first before comparing the data withsamples of a different run. Reference samples with a more or lessestablished relative gene copy numbers, are preferably included in eachexperiment to act as reference.

Validation with KIR Typed DNA Samples

The specificity of the KIR probes was verified by testing 11EBV-transformed cell lines, which were KIR-genotyped by the 10^(th)International Histocompatiblity Workshop (IHW) (Cook et al, 2003). Thecell lines were KIR-genotyped using PCR-SPP and PCR-SSOP and werecarried out in three separated laboratories. The cell lines were notgenotyped for the genes KIR2DL5A, KIR3DL3, KIR2DP1 and KIR3DP1 and alsocontained no negative controls for the genes KIR2DL1, KIR2DL4, KIR3DL1,KIR3DL2 and KIR2DS4.

In addition, DNA samples from 5 individuals were genotyped by PCR-SSPfor further verification. These 5 samples were also genotyped for thegenes KIR3DL3 and KIR3DP1 and found to contain true negative genotypicresults for KIR2DL1 and KIR2DP1. The results of the verification of thetwo probe sets are shown in tables 11-14.

Probe Set 1

KIR genotyping with probe set 1 was found to be consistent with the10^(th) IHW on 10 of the cell lines for the probes 2DL1-5, 2DS1, 2DS3-5,3DL1-2 and 3DS1. All cell lines were typed positive for the genesKIR2DP1, KIR3DP1 and KIR3DL3, the first has a frequency between 94-100%(table 9) and the last two are framework genes that are always present.Typing of the 5 individuals yielded the same results as with thePCR-SSP, except for the probe 2DS2.

Probes for 2DL5A (Same Probe in Probe Set 2)

Most studies on KIR genotyping detect the presence of KIR2DL5 and do notdifferentiate this gene between the two genes KIR2DL5A and KIR2DL5B.These two genes show a nucleotide sequence difference of only 1%. Wewere unable to design a probe for KIR2DL5B, because a specific ligationsite to discriminate KIR2DL5B from KIR2DL5A and the other KIR genes wasnot found. The probes that were designed for KIR2DL5A also detect theallele KIR3DP1*004 (table 10), because this allele contains no otherdifference in the sequence within the probe's range, thus the probe setsdo not contain specific probes for the selective detection of KIR2DL5A.In fact, KIR3DP1*004 is non-expressed, and forms a hybrid of thepromoter of KIR2DL5A and the coding region of KIR3DP1. When probe 2DL5Agenerates a signal in the MLPA, this could indicate the presence of bothKIR2DL5A and KIR3DP1*004 or either 2KIRDL5A or KIR3DP1*004 alone.However, probe 2DL5 detects the same KIR2DL5A alleles as probe 2DL5A.When probe 2DL5 is not binding and probe 2DL5A is, the absence ofKIR2DL5A and the presence of KIR3DP1*004 is demonstrated. This isclearly demonstrated by the cell lines JVM, SAVC, JBUSH, BM16, TAB089,KAS116 and the individuals 33_(—)8025 and 33_(—)8588 (FIG. 10).

Probe Set 2

Probe set 2 contains a smaller proportion of probes. A higher proportionof the probes had overlapping sequences and seven out of the ten KIRprobes needed a 10-fold higher concentration than the others to obtainpeak intensities above 500 AU.

Probe 2DS5 and 3DS1

Probes 2DS5 and 3DS1 bound to all samples including to those genotypednegative for KIR2DS5 and KIR3DS1, indicating unspecific ligation of theprobes. Probes 2DL5 and 3DS1 were not based on primer sequences usedbefore, the probe search tool on the KIR database and BLAST resultsshowed no match with other KIR genes and these probes were considered tobe specific for KIR2DS5 and KIR3DS1. No explanation could be found, whythese probes gave false positive results. These probes were excludedfrom probe set 2.

Probe 2DS1

Three out of the six negative cell lines for KIR2DS1 were typed positiveby this probe, while the two negatives from the PCR-SSP-typedindividuals were correctly typed. Probe 2DS1 target is on an intron andonly little information about intron sequences is available. The factthat other KIR genes may possess the same sequence at this position,cannot be excluded and therefore this probe is not included in the probeset.

Probe 3DP1

The probe 3DP1 in probe set 2 detects a deletion of exon 2, this alleleof KIR3DP1 is designated as KIR3DP1*003 and has a frequency of 0.72 inthe Caucasian population. Sample 33_(—)8588 of the PCR-SSP typedindividuals was typed negative for KIR3DP1 bp the MLPA probe andpositive by PCR-SSP (table 14). The conflicting typing results betweenthese two methods can be explained by the presence of exon 2 in thissample.

Cell Line LBUF

Both probe sets have genotyped this cell line positive for KIR2DL3 andnegative for KIR2DL5 and KIR2DS. In addition, probe set 1, typed LBUFnegative for KIR2DS1, KIR2DS5 and KIR3DS1 (table 11 and 13). It isreasonable to assume that the cell line LBUF that was tested, was notthe same as published before by the 10^(th) IHW. LBUF had beenKIR-genotyped by Hsu et al. 2002 (Hsu et al, 2002) and their typing wasconsistent with ours. Moreover, LBUF and the other cell lines wasKIR-genotyped with the standard PCR-SSP method and these resultsconfirmed our findings with MLPA, including the positive typing resultsof the genes KIR3DL3, KIR2DP1 and KIR3DP1 on all 11 cell lines.

Quantification of Gene Copy Numbers

For the verification of gene copy number quantification, samples with awell-defined number of copies of KIR genes were needed. Since these arenot available, we used the KIR reference panel I for this purpose,comprising 12 families of two parents and two children each. These 48reference samples have been KIR-genotyped by 15 different laboratorygroups utilizing PCR-SSP and PCR-SSOP. The Centre de'Etude duPolymorphism Humain (CEPH), Foundation Jean Dausset, Paris, France(www.cephb.fr), had prepared lymphoblastoid cell lines (LCLs) of thesefamilies. The International Histocompatibility Working Group (IHWG) Celland DNA Bank has made this panel available for commercial use(www.ihwg.org).

All the samples have been identified for the presence or absence of 16of the KIR genes and for two variants of KIR3DP1, (KIR3DP1*003 andKIR3DP1v) and two variants of KIR2DS4 (KIR1D alias KIR2DS4*003 andKIR2DS4) (table 4). Whereas, KIR3DP1 of the KIR reference panel I ischaracterized by the absence of exon 2 and the KIR3DP1v indicates theremaining KIR3DP1 alleles. KIR1D contains a 22-bp deletion in Ig-likedomain D2, causing a frame shift and early stop codon which lead to atruncated protein product (Hsu et al, 2002).

The haplotypes of these six families were also available as shown inFIG. 7. In addition this figure shows the pedigrees of the 12 families.Because of the information about the haplotypes, we could assume thatsome samples exhibit at least two copies of KIR genes. The inheritancepatterns of these copy numbers was deduced from the pedigreeinformation. The reference panel has at the same time been utilized asan extra verification step for the specificity of the probes.

Specificity in KIR Genotyping

With both probe sets difficulties were experienced with generatingreliable data of the MLPA experiments with the KIR reference panel,presumably this is caused by the lower quality of the DNA samples, asthis did not occur with the genomic DNA samples of the previousexperiments. The DQ values of the control probes had a higher frequencyoutside the proposed normal range [0.8-1.2]. Therefore, data of a numberof samples is missing and these samples should be tested in the future.

Probe Set 1

16 probes: 2DL1-5A, 2DS1, and 2DS3-5, 3DL1-3, 3DS1, 2DP1 and 3DP1 weretested and the majority of the probes genotyped the KIR reference panelaccordingly to what has been reported, except there were somedifferences with probes 2DP1 and 2DL5. These samples were correctlytyped by probe set 2.

Probe Set 2

The probes: 2DL1-5A, 2DS2, 2DS4, 3DL1-3, 3DS1, 2DP1 and 3DP1, in total14 probes were tested on the reference panel. Probe 3DP1 was designedfor KIR3P1*003 (denoted as 3DP1 in table 4) and its specificity for thisallele was confirmed with the reference panel. Probe 2DL2 typedapproximately 58% false positive and probe 2DL1 typed three of the fournegative of the panel to be positive and, therefore, no further testinghas been done with these two probes. Probe 2DS2 typed around 15%incorrectly as negative, although in a previous run which was rejectedbecause of the DQ values of the controls, these two samples were typedpositive. These samples need to be revised before a conclusion aboutprobe 2DS2 can be drawn. Probe 2DS4 gave one false negative result(sample 1333-8281). Only 80% of the KIR2DS4 alleles can be detected bythis probe because of a gene variant that is 4 bases away from theligation site in 1 out of 9 alleles. The right part of this probe willbe redesigned with an UIB code on this position.

Quantification of CNV

Probes that have been demonstrated to be accurate in KIR genotyping inboth probe sets have been analyzed for their ability in copy numberquantification. Relative quantification of CNV with one probe is simplynot reliable because gene variations near the ligation site of the probemay influence the outcome in DQ value. This is especially true for KIRsequences, because they show a high level of gene variation, whiledemonstrating a homology up to 99%. Certain probes discriminate thedifferent KIR genes only by one nucleotide difference at their ligationsite. A gene variant near the ligation site of the target gene may leadto a lower probe signal. Alternatively, a gene variant at one of theother KIR genes might cause a probe to recognize this gene as itstarget, thus enhancing the probe signal. Therefore only the KIR genes ofthe families with the reported haplotype and the complete MLPA data ofthe two probes are analyzed for copy numbers.

The DQ values of the control probes of both probe sets on each samplewere compared to check if the MLPA data are reliable. The nine controlprobes should generate the same DQ values as these control probes arethe same in both probe sets and are tested on the same sample. Sampleswith less than seven comparable control probe DQ values between the twoprobe sets were excluded. Next, the DQ values of the KIR probes wereevaluated. We interpreted the following; DQ values of 0.3< as 0 copiesof that gene, DQ [0.4-0.7]=1 copy, DQ [0.8-1.2]=2 copies, DQ [1.3-1.7]=3copies, DQ [1.8-2.2]=4 copies, DQ [2.3-2.7]=5 copies, etc. Theborderline values, such as a DQ of 0.7 are questionable and when thesecond probe obviously quantified 1 copy of this gene, 0.7 wasconsidered as 1 copy, the same approach is applied with other borderlinevalues.

FIGS. 11A and 12A show the pedigrees of the families 1347 and 1349,respectively and the legends for the haplotype are displayed below. Thecopy numbers of the KIR genes are listed in the FIGS. 11A and 12A nextto the pedigrees.

A difference in the quantification of the exact copy numbers wasobserved with the probes for KIR3DP1 in samples: 1347-8445, 1347-8436and 1349-8398. Probe set 1 seems to detect more copies of this gene thanprobe set 2, which is in agreement with their design. Probe 3DP1 (1)detects all the KIR3DP1(v) alleles and probe 3DP1 (2) detects onlyKIR3DP1*003 denoted in the legend as 3DP1, which exhibit the exon 2deletion. The probes 2DL3 and 2DL4 in probe set 1 detected fewer copiesnumbers than their counterparts in probe set 2. Probe 2DL3 and probe2DL4 might have problems with the presence of gene variants at theirtarget sequence, whereas these probes in probe set 2 have no genevariants in the probe target sequence and give a coverage of 100% (table10). The probes for KIR3DL1 quantified the members of family 1349differently. The probe in probe set 1 covers different alleles than theprobe in probe set 2, the coverage rate are 78% and 41% respectively dueto gene variants present at their target sequence more then 10 basesaway from the ligation site, that might influence the binding efficiencyand thereby the peakhights. Also here adding IUB codes in the probesequence will overcome the problem of misinterpretation of copy numberdifferences between individuals.

Despite the differences in copy number quantification of a number ofprobes, the overall inheritance pattern of the gene copies was inagreement with the inheritance of the haplotypes. For example the fourframework genes KIR3DL3, KIR3DP1, KIR2DL4 and KIR3DL2 were present inall samples and at least 2 copies of each of these genes have beenfound. This indicates that these genes are present in at least one copyat each allele and are inherited from both parents. Examination offamily 1347 revealed that the father, haplotype a/b (sample 8440) hasthree copies of gene KIR2DL5 on one allele, haplotype b and one on theother, haplotype a and has past haplotype b, with the three copies tothe child (sample 8436) and the allele haplotype a, with one copy to theother child (sample 8412). For the family 1349, one copy of KIR2DS4 isbelieved to reside on one allele, haplotype c and two on the other,haplotype d of the mother (sample 8399). Because both children,haplotype b/c and haplotype a/c (sample 8393 and 8636), respectively,inherited the allele with two copies from their mother as they have boththe haplotype c and one child (sample 8636) inherited one copy of thisgene from its father, haplotype a. Also when the inheritance patterns ofthe remaining copy numbers of genes were analyzed, no inconsistency withthe inheritance patterns of the haplotypes could be found. The rest ofthe families with fully reported haplotypes should be tested again toobtain complete data of all the members within one family, before theinheritance patterns and copy numbers can be analyzed.

Discussion

Before the present invention, the main problem in designing syntheticMLPA probes for KIR genotyping was to design probes specific enough forthe target gene, but still sensitive enough to detect most of thealleles present in the population. KIR genes have very high level ofhomology (85-99%) in the sequences of both exons and introns and show anextensive degree of gene variation.

The MLPA is a good method, because it can discriminate target sequencesthat only differ one nucleotide at the ligation site. The presentinventors designed synthetic MLPA probes consisting of three probe partswhich added a second ligation site, so that an extra discriminationpoint was provided. In addition these three-part probes made it possibleto elongate the ligated probe size, the longest probe tested in thisstudy was 223 bp (Ctr 10). Due to the better quality of the probes andthree-part probes, the number of probes in a synthetic MLPA probe setaccording to the invention is less restricted by the size of the ligatedprobes.

This study has demonstrated that the MLPA with two synthetic probe setsis reliable in KIR genotyping, as these two probe sets have been wellvalidated by three independent approaches. The two probes setscomplement each other in the detection and coverage of the KIR alleles,which yielded in no false negatives any more in all the samples used forverification. Even after exclusion of the probes that may have generatedfalse positives from the probe sets, all 16 KIR genes can still beconsistently detected for their presence or absence. This makes the MLPAmethods used in this Example in a qualitative sense comparable to thePCR-SSP and PCR-SSOP methods. However time and work is saved with theperformed Example, as only two reactions are needed to generate acomplete KIR-genotype profile.

In summary, probe set 1 contains the probes 2DL1-5, 2DS1, and 2DS3-5,3DL1-3, 3DS1, 2DP1 and 3DP1, in total 15 probes. Probe set 2 containsthe probes 2DL3-5, 2DS2-4, 3DL1-3, 2DP1 and 3DP1, in total 11 probes.Together these two probe sets are accurate for the typing of 16 KIRgenes and for quantifying relative copy numbers of at least 9 KIR genes.

Example 2

This Example presents additional probes for KIR genotyping and copynumber variation analysis with multiplex ligation dependent probeamplification (MLPA). Here, probes are presented for all 17 KIR genesKIR2DL1-5, KIR2DS1-5, KIR3DL1-3, KIR3DS1, KIR3DP1 and KIR2DP1, includingKIR2DL5a and KIR2DL5b, KIR3DP1v and several null alleles. The extendedprobesets 1 and 2 are listed in FIGS. 3C and 3D, respectively. As inexample 1, the specificity of the probes was validated by comparison ofthe samples for the KIR genotypes obtained with PCR-SSOP and PCR-SSPmethods, and the ability of the probes to quantify relative gene copynumbers was examined with 12 families, each consisting of two parentsand two offspring, which have been genotyped for most KIR alleles.

Materials & Methods

For DNA selection/isolation, probe design, MLPA reaction,electrophoresis and analysis according to materials & methods of example1 with the exception that no competitors were used and data werenormalized with Soft genetics Genemarker v1.85, using internal controlprobe normalization (http://www.softgenetics.com/papers/MLPA) andsynthetic references.

Results Extended Probesets

With the extended probesets 1 and 2 all KIR genes and several KIR genevariants were detected.

The extended probe set 1 depicted in FIG. 3C detects the same genes asprobe set 1 of example 1 but additional probes are added and thereforeadditional KIR gene variants are now detected. Additional probes thatare added are 2DL5B, 2DL4N (2DL4*007,008,009,011), 3DL1*024N.

The extended probe set 2 as depicted in FIG. 3D detects the same genesas probe set 2 of example 1 but additional probes are added andtherefore additional KIR gene variants are now detected. Additionalprobes that are added are 2DL5B, 3DS1*049N and 2DS4N (2DS4*004, *006,*007,*008 and *009). KIR2DS4N is also called KIR1D.

Probe 3DP1

The probe 3DP1 in extended probe set 2 detects a deletion of exon 2,this allele of KIR3DP1 is designated as KIR3DP1*003, KIR3DP1*005 orKIR3DP1*006.

Probes for 2DL5A and 2DL5B

With the extended probesets 1 and 2 KIR2DL5A and 2DL5B are now alsodetected. The probes that were designed for KIR2DL5A and KIR2DL5B alsodetect the alleles KIR3DP1 variants (table 10, KIR3DP1v). When probe2DL5A or 2DL5B generates a signal in the MLPA, this could indicate thepresence of both KIR2DL5A and KIR3DP1v or KIR2DL5B and KIR3DP1vrespectively. Alternatively, when probe 2DL5A or 2DL5B generate a signalin the MLPA the presence of either KIRDL5A or KIR3DP1v alone (with probe2DL5A) or KIR2DL5B or KIR3DP1v alone (with probe 2DL5B) is indicated.Thus with these probes 2DL5A and 2DL5B more than one KIR gene isdetected. Therefore, these probes are not suitable to determine copynumber variation (see FIG. 13).

Copy Number Variation (CNV)

For all KIR alleles except KIR3DP1 variants (KIR3DP1v), KIR2DL5A and2DL5B copy number variation is determined with extended probesets 1 and2 (FIG. 13).

Quantification of CNV

A difference in the quantification of the exact copy numbers as comparedto example 1 was elaborated by studies with the extended probesets.Optimization of the probe set initially used in FIG. 11A, has nowresulted in a 100%-perfect match with the validated KIR data in the inexample 1 genotyped pedigrees. None of the MLPA probes gave afalse-positive or false-negative signal in the 10^(th) ICW familiestested as exemplified by the analysis of families 1347 and 1349 (FIGS.11B & 12B). Thus, both probe set 1 and/or 2 and extended probe sets 1and/or 2 are suitable for detection of KIR genes and for determinationof relative copy number variation, but extended probe sets 1 and/or 2,as depicted in FIGS. 3C and 3D, are preferred.

Specificity and Quantification for KIR Haplotyping

From the MLPA data within pedigrees haplotyping can be inferred. Firstof all, the framework genes KIR3DL3 and KIR3DP1 for the first block inboth haplotypes A and B (FIG. 6) and KIR2DL4 and KIR3DL2 are present ina fixed copy number of 2 genes. However, KIR3DP1 may be present asso-called KIR3DP1v variant (see also FIG. 7, grey boxes represent theframework KIR genes in both haplotypes A and B). In case of haplotype Bthe presence of KIR genes may vary widely (FIG. 6), making thishaplotype an important contribution to the variation within the KIR genecluster.

In family 1347, we have deduced, using the extended probesets, from thepedigree a correct and complete KIR haplotype analysis (FIG. 11B). Atthe single gene level the MLPA results offers insight into the patternsof inheritance. The sibs inherited from their parents different KIRhaplotypes, which—for instance—resulted in the variation in KIR2DL5 genecontent. Thus, both sibs have 2 of these genes, containing 2 KIR2DL5genes from the father (who carries 4 KIR2DL5 genes in total) and onenull-haplotype from the mother. From the present data from theliterature or the current MLPA data, it cannot yet be distinguishedwhether the two KIR2DL5 genes that both sibs have inherited, are thesame alleles, or whether the KIR2DL5 are located in the first or secondblock of the so-called B haplotype (see also FIG. 6).

At the haplotype level, patterns of inheritance are deduced for theremaining non-framework KIR genes in this pedigree, e.g. KIR2DL3,KIR2DS2, KIR2DL2, KIR2DP1, and KIR2DL1 genes in the first block ofhaplotype B, generally located in between the framework genes KIR3DL3and KIR3DP1 genes (see also FIG. 6).

In case of the first block of haplotype B, the results are explained bythe inheritance of a KIR2DL3-KIR2DP1-KIR2DL1 haplotype from the fatherand the KIR2DS2-KIR2DL2-KIR2DP1-KIR2DL1 haplotypic block from themother. In case of the second block of haplotype B, it is clear that theKIR3DS1-KIR2DS3-KIR2DS1 haplotype has been inherited from the father andthe KIR3DL1-KIR2DS4 from the mother. Yet, one sib (8436) must have losta KIR3DL1 gene according to our MLPA analysis. Sib 8436 has the normal3DL1 present in our MLPA, though sib 8412 has inherited a 3DL1N variantgene in stead of the normal 3DL1 gene. This is just by normalinheritance so not an exception.

SSP-PCR can not discriminate between 3DL1 variants (also not between3DS1 variant genes nor 2DL4 variant genes).

At the haplotype level, patterns of inheritance are similarly deducedfor the pedigree of family 1349 (FIG. 12B). Apart from the framework KIRgenes in this pedigree, the non-framework genes form the haplotype Bthat are inherited “en bloc”.

In case of these two sibs, 1349-8393 and -8636, the KIR variation can bewell explained by inheriting different KIR haplotypes from both parents.

With respect to the first block of haplotype B, the results areexplained by the inheritance of one of his two similarKIR2DL3-KIR2DP1-KIR2DL1 alleles from the father and one from the mother(while this female also carried a smaller KIR2DL3-KIR2DP1 haplotypicblock).

In case of the second block of haplotype B, it is clear that the fathercarries a KIR3DL1-KIR2DS4 combination on one allele and a separateKIR2DS3-KIR2DS4-KIR2DS1 haplotypic on the other allele that weredifferently inherited by the two sibs, whereas the mother carries twoidentical KIR3DL1-KIR2DS4 alleles.

In FIGS. 11 and 12 the standard SSP PCR results are compared with ourMLPA data with the extended probe sets 1 & 2 for the pedigrees in theCEPH families 1347 and 1349.

Two KIR haplotype models have been described (see for instance: H. Li,PLoS Genetics, 2008, 4, 11:e1000254; M. Uhrberg, Eur. J. Imm.Highlights, 2005, 35:10-15; M. Carington, The KIR Gene Cluster, 2003; K.Hsu, Imm. Reviews, 2002, 190:40-52). The conventional KIR haplotypemodel assumes that there are two haplotypes A and B. Both haplotypes Aand B contain the framework genes 3DL3, 3DP1, 2DL4, and 3DL2. Then thereare the KIR genes 2DP1, 2DL1 and 2DS4 that are common for bothhaplotypes, but only the haplotype A contains 2DL3, 3DL1 and 2DS4.Haplotype B is more variable and can contain the KIR genes 2DS1, 2DS2,2DS3, 2DS4, 2DS5, 3DS1, 2DL2 and 2DL5 (apart form the aforementionedframework genes). In more than 96% of the worldwide global populationthe A haplotype at KIR gene cluster contains the KIR genes 3DL3, 2DL3,2DP1, 2DL1, 3DP1, 2DL4, 3DL1, 2DS4 and 3DL2 (see also:www.allelfrequencies.net).

The novel KIR haplotype model assumes that haplotype A and B are presenton the two different chromosomes. Therefore any individual can representan AA, AB or BB genotype. Based on the genes that are present in the DNAsample of that individual, one can conclude which haplotypes are presentand the positive genes from the assay can be divided over bothhaplotypes according to the rules that certain KIR genes are presentonly in one of the haplotypes A or B, essentially as was mentionedabove.

For the SSP PCR data the two haplotype models are shown to interpretpossible CNV results, resp. the conventional KIR haplotype model in FIG.11B1 and 12B1 and the novel KIR haplotype model in FIG. 11B2 and 12B2.FIG. 11B3 and 12B3 show the results of our MLPA data with the extendedprobe sets 1 & 2 compared with both the SSP PCR data according to theconventional KIR haplotype model and with the novel KIR haplotype model.

In conventional KIR haplotype model in FIGS. 11B1 and 12B1 the KIR generegion is described by framework genes (3DL3, 3DP1, 2DL4 and 3DL2),genes that can be present in both A and B haplotypes (2DP1, 2DL1 and2DS4) and haplotype-specific genes. The KIR genes 2DL3, 3DL1 and 2DS4are specific for haplotype A. while the KIR genes 2DL5, 2DS1, 2DS2,2DS3, 2DS5, 3DS1 and 2DL2 are specific for haplotype B. The haplotype Ais constant to a high degree. In more than 96% of the global populationhaplotype A consists of 3DL3, 2DL3, 2DP1, 2DL1, 3DP1, 2DL4, 3DL1, 2DS4and 3DL2 (www.allelefrequencies.net). Haplotype B is more variable andcarries more activating KIR genes.

FIGS. 11B2 and 12B2 show the interpretation for the respective familiesbased on the novel KIR haplotype model and SSP-PCR data from CEPH-IHWG.

FIGS. 11B3 and 12B3 show the copy number variation for the respectivefamilies. In table 3 Copy number variation of KIR genes by MLPA isdetermined by 2 probes for each gene, except for the N-variant genes(single probe detection by definition), including those genes marked byan asterisk.

For the 3DP1v gene variant a combination of 3 probes has been designed.CNV can be deduced from a comparison between the results for the probesfor 2DL5, 2DL5a and 2DL5b.

The 2DS4N KIR probe is designed to detect the KIR-2DS4 deletion-variantgenes *003 to *009, while SSP-PCR only detects 2DS4 variant *003(designated 1D).

In FIG. 12B3 KIR3DP1 variants are detected using MLPA (table 3), whereasKIR3DP1 variants are not detected when SSP-PCR is used. SSP-PCR ofKIR3DP1v results in a band of 1672 bp that is obtained from the 3DP1gene. Because this is a large fragment which are known to be difficultto detect. Therefore, a DNA sample can be positive for KIR3DP1v whenMLPA is used but appear to be negative for KIR3DP1v when SSP-PCR isused.

CONCLUSION

Extended probe set 1 contains the probes 2DL1-5, 2DS1-5, 3DL1-3, 3DS1,2DP1 and 3DP1, in total 20 probes. Extended probe set 2 contains theprobes 2DL1-5, 2DS1-5, 3DL1-3, 3DS1, 2DP1 and 3DP1, in total 20 probes.Together these two probe sets are accurate for the typing of all 17 KIRgenes, and 7 variant KIR gene variants (i.e. 2DL5a, 2DL5b, 3DP1v, andthe null-variants 2DL4N, 3DL1N, 3DS1N, and 2DS4N), and for quantifyingrelative copy numbers of at least all 17 different KIR genes, and 4null-variant (2DL4N, 3DL1N, 3DS1N, and 2DS4N) (see FIG. 13).

Example 3

The advantage of probe sets comprising three probe parts according tothe present invention is that at least two different SNPs can bedetected with one probe set. For instance, in a probeset consisting ofthree probe parts two sites for ligation are preferably present. A leftprobe part and middle probe part are ligated and additionally a middleprobe part and right probe part are ligated. At each ligation site a SNPcan be detected. With conventional MLPA probe sets, consisting of twohalf probes, only one SNP can be detected per probe set, because onlyone site for ligation is present.

In this Example detection of the Null allele of KIR3DL1 with a probesetconsisting of three probes (one left probe part, one middle probe partand one right probe part) is described. This example is illustrated inFIG. 1C.

Materials & Methods

The null allele, called KIR3DL1*024N, is discriminated from KIR3DL1using three probes of the invention. Partial probes (probe numbers asdepicted in FIG. 3C) used in this example are:

711A - KIR3DL1 WT Left probe part:5′-PO4 GGTTCCCTAAGGGTTGGACCCCTCACGCCTCGTTGGACA-3′711D - KIR3DL1*024N Left probe part:5′-PO4-GGGTTCCCTAAGGGTTGGACAAGGACCCCTCACGCCTCGTTGG AC-3′711B - KIR3DL1 Middle probe part:5′-PO4-GATCCATGATGGGGTCTCCAAGGCCAATTTCTCCATCGGTCCC ATGATGCT-3′711C - KIR3DL1 Right probe part:5′-PO4-GCCCTTGCAGGGACCTACAGATGCTACGGTTCTGGTCTAGATT GGATCTTGCTGGCAC-3′

For DNA selection/isolation, probe design, MLPA reaction,electrophoresis and analysis see materials & methods of example 1.

With these partial probes 2 probe sets can be formed. Those two probesets consist of different left probe parts, but share the middle andright probe parts.

Results and Discussion

The final base of middle probe part 711B is a thymine. This thymine isspecific for KIR3DL1 genes while all other KIR genes have a differentbase at this position. Therefore, with probe part 711B KIR3DL1 isdiscriminated from other KIR genes. Ligation between the middle probepart (711B) and right probe part (711C) will only occur when KIR3DL1genes are present. The final base of left probe part 711A is an adenine.This base is present in wildtype KIR3DL1 gene but deleted in the KIR3DL1null allele, KIR3DL1*024N. Thus, probe part 711A containing an adenineat the final base position is specific for the wildtype KIR3DL1 gene andligation between the 711A left probe part and the middle probe part(711B) will only occur if the KIR3DL1 wildtype gene is present. In leftprobe part 711D the final adenine is removed. Thus, probe part 711D isspecific for null allele KIR3DL1*024N and ligation between the 711D leftprobe part and the middle probe part (711B) will only occur ifKIR3DL1*024N is present.

Thus these two probe sets each detect 2 SNPs, namely those SNPs that arespecific for KIR3DL1 wildtype gene and null allele KIR3DL1*024N becauseboth the left probe part and the middle probe part are SNP-specific.

TABLE 1 KIR genes and proteins names, adapted from KIR Nomenclaturereport 2002 (Marsh et al, 2002). Gene Protein symbol symbol AliasesKIR2DL1 KIR2DL1 cl-42, nkat1, 47.11, p58.1, CD158a KIR2DL2 KIR2DL2cl-43, nkat6, CD158b1 KIR2DL3 KIR2DL3 c1-6, nkat2, nkat2a, nkat2b, p58,CD158b2 KIR2DL4 KIR2DL4 103AS, 15.212, CD158d KIR2DL5A KIR2DL5AKIR2DL5.1, CD158f KIR2DL5B KIR2DL5B KIR2DL5.2, KIR2DL5.3, KIR2DL5.4KIR2DS1 KIR2DS1 EB6ActI, EB6ActII, CD158h KIR2DS2 KIR2DS2 cl-49, nkat5,183ActI, CD158j KIR2DS3 KIR2DS3 nkat7 KIR2DS4 KIR2DS4 cl-39, KKA3,nkat8, CD158i KIR2DS5 KIR2DS5 nkat9, CD158g KIR2DP1 KIR2DP1 KIRZ, KIRY,KIR15, KIR2DL6 KIR3DL1 KIR3DL1 cl-2, NKB1, cl-11, nkat3, NKB1B, AMB11,KIR, CD158e1 KIR3DL2 KIR3DL2 cl-5, nkat4, nkat4a, nkat4b, CD158k KIR3DL3KIR3DL3 KIRC1, KIR3DL7, KIR44, CD158z KIR3DS1 KIR3DS1 nkat10, CD158e2KIR3DP1 KIR3DP1 KIRX, KIR48, KIR2DS6, KIR3DS2P, CD158c

TABLE 2 Number of currently known alleles for each KIR gene and thedifferent protein products they encode (IPD KIR database,http://www.ebi.ac.uk/ipd/kir). Gene 2DL1 2DL2 2DL3 2DL4 2DL5 2DS1 2DS22DS3 Alleles 25 11 9 25 21 12 12 9 Proteins 28 7 8 12 11 8 6 3 Gene 2DS42DS5 3DL1 3DS1 3DL2 3DL3 2DP1 3DP1 Alleles 20 12 52 14 45 55 5 8Proteins 13 9 46 12 40 31 0 0

TABLE 3 KIRs and their cognate ligands (Carrington et al, 2003;Middleton et al, 2005; Du et al, 2007). The ligands of the other KIRsare unknown or uncertain. Inhibitory KIRs Ligands Activating KIRsLigands 2DL1 HLA-C group 2, 2DS1 HLA-C group 2 allotypes allotypes Cw1,4, 5, 6, 17, 18 Cw1, 4, 5, 6, 17, 18 2DL2 and 2DL3 HLA-C group 1, 2DS2HLA group 1, allotypes allotypes Cw1, 3, 7, 8, 13, 14 Cw1, 3, 7, 8, 13,14 2DL4 HLA-G 2DS4 HLA-C 3DL1 HLA-B, Bw4 3DS1 HLA-B, Bw4 3DL2 HLA-A3 andA11 allotypes

TABLE 4 The KIR Reference Panel I from the IHWG(http://www.ihwg.org/cellbank/dna/refpan_nkkir_table.html). 2DS4indicates all alleles except KIR2DS4*003 and 1D indicates onlyKIR2DS4*003. 3DP1 indicates KIR3DP1*003 (deletion of exon 2) only and3DP1v indicates all alleles except KIR3DP1*003

Note: “1” = presence of KIR gene “0” = absence of KIR gene shaded cells(N = 16) represent four informative families selected for the Phase IIreference panel

TABLE 5 The 17 KIR probes that have been designed and tested for probeset 1. The size of the complete MLPA probe and the size of the separateprobe parts and the concentration used are listed in this table. SizeProbe Size Concentration Code Probe [bp] Part [bp] (fmol) 420A 2DL2 96Left 48 1 420B Right 48 512A 3DL3 100 Left 50 1 512B Right 50 540A 2DS3108 Left 54 10 540B Right 54 404A 3DL2 112 Left 56 1 404B Right 56 405A2DP1 121 Left 65 1 405B Right 56 406A 3DP1 125 Left 66 1 406B Right 59504A 2DS4 137 Left 61 1 504B Right 76 408A 2DL5 142 Left 57 1 408BMiddle 32 408C Right 53 514A 3DL1 149 Left 74 1 514B Right 75 526A 2DS2154 Left 57 1 526B Middle 34 526C Right 63 507A 2DL5A 165 Left 66 1 507BMiddle 32 507C Right 67 419A 2DL4 170 Left 59 1 419B Middle 54 419CRight 57 528A 2DS5 185 Left 67 1 528B Middle 47 528C Right 71 413A 2DL1189 Left 72 1 413B Middle 64 413C Right 53 416A 2DS1 195 Left 78 10 416BMiddle 67 416C Right 50 415A 2DL3 213 Left 75 10 415B Middle 69 415CRight 69 418A 3DS1 218 Left 81 10 418B Middle 64 418C Right 73

TABLE 6 The 17 KIR probes that have been designed and tested for probeset 2. The size of the complete MLPA probe and the size of the separateprobe parts and the concentration used are listed in this table. SizeProbe Size Concentration Code Probe [bp] Part [bp] (fmol) 543A 2DS1 96Left 48 10 543B Right 48 544A 2DS2 100 Left 50 1 544B Right 50 537A 2DL5108 Left 54 1 537B Right 54 513D 2DS3 112 Left 52 10 513B Right 60 518A3DP1 121 Left 61 1 518B Right 60 542A 2DP1 125 Left 60 1 542B Right 65541A 3DS1 134 Left 67 10 541B Right 67 524A 2DS4 137 Left 66 10 524BRight 71 545A 2DS5 144 Left 68 10 545B Right 76 409A 3DL1 149 Left 60 10409B Middle 34 409C Right 55 506A 3DL3 154 Left 54 10 506B Middle 48506C Right 52 507A 2DL5A 165 Left 66 1 507B Middle 32 507C Right 67 539A2DL2 170 Left 60 1 539B Middle 46 539C Right 64 525A 2DL1 190 Left 64 10525B Middle 62 525C Right 64 538A 3DL2 r 195 Left 70 1 538B Middle 60538C Right 65 417A 2DL3 213 Left 75 10 417B Middle 69 417C Right 69 517A2DL4 218 Left 73 10 517B Middle 68 517C Right 77

TABLE 7 The control probes used in the two probes sets. The size of thecomplete MLPA probe and the size of the separate probe parts and theconcentration used for the probe sets are listed in this table. SizeProbe Size Concentration Code Probe (Gene) [bp] part [bp] (fmol) 201Ctrl 2 (FGF3) 92 Left 45 1 Right 47 202 Ctrl 3 (BCAS4) 104 Left 52 1Right 52 203 Ctrl 4 (LMNA) 116 Left 58 1 Right 58 204 Ctrl 5 (PARK2) 130Left 44 3 Middle 41 Right 45 205 Ctrl 7 (MSH6) 160 Left 59 1 Middle 42Right 59 206 Ctrl 8 (GALT) 175 Left 58 1 Middle 59 Right 58 207 Ctrl 9(SPG4) 180 Left 60 1 Middle 60 Right 60 210 Ctrl 1 (IL-4) 208 Left 73 1Middle 69 Right 66 209 Ctrl 10 (NF2) 223 Left 78 1 Middle 69 Right 76

TABLE 8 The competitors of the control probes. The size of thecompetitor, the part of the control probes used and concentration usedfor the probe sets are listed in this table. code gene length [bp] probepart Concentration (fmol) 201X Ctrl 2 (FGF3) 30 Left 10 202X Ctrl 3(BCAS4) 30 Left 10 203X Ctrl 4 (LMNA) 30 Left 3 205X Ctrl 7 (MSH6) 50Left 0 207X Ctrl 9 (SPG4) 50 Left 1

TABLE 9 KIR gene frequencies in the Caucasian population. Thefrequencies are derived from several studies performed worldwide in theCaucasian population and are available on www.allelfrequencies.net.KIR2DL1 KIR2DL2 KIR2DL3 KIR2DL4 KIR2DL5 KIR2DS1 KIR2DS2 KIR2DS3 88-100%39-63% 57-94% 100% 36-61% 27-49% 25-63% 19-42% KIR2DS4 KIR2DS5 KIR3DL1KIR3DL2 KIR3DL3 KIR3DS1 KIR2DP1 KIR3DP1 87-98% 21-46% 76-98% 99-100%99-100% 26-50% 94-100% 97-100%

TABLE 10 KIR alleles detected by the probes and the coverage of thetotal KIR alleles, except for 3DP1v, by probe sets 1 and 2, as depictedin FIG. 3A and 3B. All KIR alleles including 3DP1v are also detected byextended probe sets 1 and 2, as depicted in FIG. 3C and 3D Coveragelower then 100% are caused by gene variants that are present in thetarget sequence to which the probes bind. The alleles shown here thatcan be detected by the probes are generated with the primer or probeblast tool on the IPD KIR database. The percentage of the total KIRalleles that can be covered by the probes is calculated by dividing thenumber of alleles for each probe by the number of total alleles that isreported on the website. Certain alleles are underlined where thecoverage of both probe sets is not 100% due to gene variants present inthe target sequence. Probe set Probe set 1 Probe set 2 1 + 2 PROBEALLELES COVERAGE PROBE ALLELES COVERAGE COVERAGE 2DL1 2DL1*0012DL1*00402 100% 2DL1 2DL1*001 2DL1*00402 100% 100% 2DL1*002 2DL1*0052DL1*002 2DL1*005 2DL1*00301 2DL1*006 2DL1*00301 2DL1*006 2DL1*00302012DL1*007 2DL1*0030201 2DL1*007 2DL1*0030202 2DL1*008 2DL1*00302022DL1*008 2DL1*00303 2DL1*009 2DL1*00303 2DL1*009 2DL1*0040101 2DL1*0102DL1*0040101 2DL1*010 2DL1*0040102 2DL1*0040102 2DL2 2DL2*001 2DL2*004100% 2DL2 2DL2*001 2DL2*003  80% 100% 2DL2*002 2DL2*005 2DL2*0022DL2*005 2DL2*003 2DL3 2DL3*001 2DL3*004  86% 2DL3 2DL3*001 2DL3*005100% 100% 2DL3*002 2DL3*005 2DL3*002 2DL3*006 2DL3*003 2DL3*006 2DL3*0032DL3*007 2DL3*004 2DL4 2DL4*00101 2DL4*00501  54% 2DL4 2DL4*001012DL4*00601 100% 100% 2DL4*00102 2DL4*00601 2DL4*00102 2DL4*006022DL4*00105 2DL4*00602 2DL4*0010301 2DL4*007 2DL4*00201 2DL4*0072DL4*0010302 2DL4*0080101 2DL4*00202 2DL4*0080101 2DL4*001042DL4*0080102 2DL4*003 2DL4*0080201 2DL4*00105 2DL4*0080103 2DL4*0042DL4*011 2DL4*00201 2DL4*0080104 2DL4*00202 2DL4*0080201 2DL4*002032DL4*0080202 2DL4*003 2DL4*009 2DL4*004 2DL4*010 2DL4*00501 2DL4*0112DL4*00502 2DL4*012 2DL5 2DL5A*0010101 2DL5B*003 100% 2DL5 2DL5A*00101012DL5B*00601  54% 100% 2DL5A*0010102 2DL5B*004 2DL5A*0010102 2DL5B*0072DL5A*0050101 2DL5B*00601 2DL5B*003 2DL5B*00801 2DL5A*0050102 2DL5B*0072DL5B*004 2DL5B*0020101 2DL5B*00801 2DL5B*0020102 2DL5B*0092DL5B*0020103 2DL5A 2DL5A*0010101 2DL5A*0050101 100% 2DL5A Same probe asin probe set 1. 100% 100% 2DL5A*0010102 2DL5A*0050102 3DP1*004  14%3DP1v 2DS1 No match found in the KIR 2DS1 No match found in the KIRdatabase. BLAST result in match database. Probe designed on withKIR2DS1v alias KIR2DS1*002 intron 6. 2DS2 2DS2*0010101 2DS2*002  90%2DS2 No match found in the KIR 90% 2DS2*0010102 2DS2*003 database. Probedesigned on 2DS2*0010103 2DS2*004 intron 2 and 3. 2DS2*00102 2DS2*0052DS2*00103 2DS3 2DS3*00101 2DS3*002 100% 2DS3 2DS3*00101 2DS3*002 100%100% 2DS3*00102 2DS3*003N 2DS3*00102 2DS3*003N 2DS3*00103 2DS3*0042DS3*00103 2DS3*004 2DS3*00104 2DS3*00104 2DS4 2DS4*0010101 2DS4*003100% 2DS4 2DS4*0010101 2DS4*003  80% 100% 2DS4*0010102 2DS4*0042DS4*0010102 2DS4*006 2DS4*0010103 2DS4*006 2DS4*0010103 2DS4*0072DS4*00102 2DS4*007 2DS4*00102 2DS4*009 2DS4*00103 2DS4*009 2DS52DS5*001 2DS5*004 100% 2DS5 2DS5*001 2DS5*004 100% 100% 2DS5*00201012DS5*005 2DS5*0020101 2DS5*005 2DS5*0020102 2DS5*006 2DS5*00201022DS5*006 2DS5*0020103 2DS5*007 2DS5*0020103 2DS5*007 2DS5*003 2DS5*0082DS5*003 2DS5*008 3DL1 3DL1*00101 3DL1*027  78% 3DL1 3DL1*00101 3DL1*021 41% 88% 3DL1*00102 3DL1*028 3DL1*002 3DL1*022 3DL1*002 3DL1*0293DL1*00401 3DL1*023 3DL1*00401 3DL1*030 3DL1*00402 3DL1*024N 3DL1*004023DL1*031 3DL1*00501 3DL1*025 3DL1*00501 3DL1*032 3DL1*00502 3DL1*0263DL1*00502 3DL1*033 3DL1*006 3DL1*027 3DL1*007 3DL1*034 3DL1*0073DL1*028 3DL1*008 3DL1*035 3DL1*008 3DL1*029 3DL1*009 3DL1*036 3DL1*0093DL1*030 3DL1*01501 3DL1*037 3DL1*01502 3DL1*038 3DL1*016 3DL1*0393DL1*01701 3DL1*040 3DL1*01702 3DL1*041 3DL1*018 3DL1*042 3DL1*024N3DL1*043 3DL1*025 3DL1*044 3DL1*026 3DL1*057 3DL2 3DL2*00101 3DL2*00902 47% 3DL2 3DL2*00101 3DL2*010  45% 61% 3DL2*002 3DL2*013 3DL2*0023DL2*011 3DL2*00301 3DL2*014 3DL2*00301 3DL2*012 3DL2*004 3DL2*0163DL2*004 3DL2*013 3DL2*005 3DL2*017 3DL2*005 3DL2*015 3DL2*00701013DL2*018 3DL2*006 3DL2*016 3DL2*0070102 3DL2*019 3DL2*0070101 3DL2*0203DL2*008 3DL2*020 3DL2*0070102 3DL2*021 3DL2*00901 3DL2*021 3DL2*0083DL3 3DL3*00101 3DL3*01102  75% 3DL3 3DL3*00101 3DL3*01303 100% 100%3DL3*00102 3DL3*012 3DL3*00102 3DL3*01304 3DL3*00103 3DL3*013013DL3*00103 3DL3*01305 3DL3*00201 3DL3*01303 3DL3*00201 3DL3*013063DL3*00203 3DL3*01304 3DL3*00202 3DL3*01307 3DL3*00204 3DL3*014013DL3*00203 3DL3*01401 3DL3*00205 3DL3*01403 3DL3*00204 3DL3*014023DL3*00207 3DL3*01405 3DL3*00205 3DL3*01403 3DL3*0030101 3DL3*0153DL3*00206 3DL3*01404 3DL3*0030102 3DL3*016 3DL3*00207 3DL3*014053DL3*00401 3DL3*017 3DL3*0030101 3DL3*015 3DL3*00402 3DL3*0183DL3*0030102 3DL3*016 3DL3*005 3DL3*020 3DL3*00401 3DL3*017 3DL3*006013DL3*021 3DL3*00402 3DL3*018 3DL3*00602 3DL3*022 3DL3*005 3DL3*0193DL3*00801 3DL3*023 3DL3*00601 3DL3*020 3DL3*00802 3DL3*024 3DL3*006023DL3*021 3DL3*00901 3DL3*025 3DL3*007 3DL3*022 3DL3*00902 3DL3*0263DL3*00801 3DL3*023 3DL3*010 3DL3*028 3DL3*00802 3DL3*024 3DL3*011013DL3*00901 3DL3*025 3DL3*00902 3DL3*026 3DL3*010 3DL3*027 3DL3*011013DL3*028 3DL3*01102 3DL3*029 3DL3*012 3DL3*030 3DL3*01301 3DL3*0313DL3*01302 3DS1 3DS1*010 3DS1*046  71% 3DS1 3DS1*010 3DS1*045  71% 86%3DS1*01301 3DS1*047 3DS1*011 3DS1*046 3DS1*01302 3DS1*048 3DS1*0123DS1*047 3DS1*014 3DS1*049N 3DS1*01301 3DS1*048 3DS1*045 3DS1*0553DS1*01302 3DS1*049N 2DP1 2DP1*00101 2DP1*0020102 100% 2DP1 2DP1*001012DP1*0020102 100% 100% 2DP1*00102 2DP1*003 2DP1*00102 2DP1*0032DP1*0020101 2DP1*0020101 3DP1 3DP1*001 3DP1*004 100% 3DP1 No matchfound on the KIR 100% 3DP1*002 3DP1*005 database. 3DP1*00301 3DP1*006Detects deletion of exon 2. 3DP1*00302

TABLE 11 Verification of KIR MLPA probe set 1 on 11 cell linesKIR-genotyped by the 10^(th) IHW. KIR genotyped Cell lines by the10^(th) IHW, results of probes set1. CODE NAME 2DL1 2DL2 2DL3 2DL4 2DL52DL5A 2DS1 2DS2 2DS3 2DS4 2DS5 3DL1 3DL2 3DL3 3DS1 2DP1 3DP1 231 JVM 1 11 1 0 4 0 1 0 1 0 1 1 4 0 4 4 240 T7507 1 1 1 1 1 4 1 1 1 1 0 1 1 4 1 44 343 OLGA 1 0 1 1 1 4 1 2 0 1 1 1 1 4 1 4 4 423 SAVC 1 0 1 1 0 4 0 2 01 0 1 1 4 0 4 4 712 JBUSH 1 0 1 1 0 4 0 2 0 1 0 1 1 4 0 4 4 723 BM16 1 01 1 0 4 0 2 0 1 0 1 1 4 0 4 4 773 LBUF

1 2 1 3 4 3 1 3 1 3 1 1 4 3 4 4 931 AMALA 1 1 1 1 1 4 1 1 0 1 1 1 1 4 14 4 1042 BM90 1 1 1 1 1 4 1 1 1 1 1 1 1 4 1 4 4 1102 TAB089 1 0 1 1 0 40 2 0 1 0 1 1 4 0 4 4 122 KAS116 1 0 1 1 0 4 0 2 0 1 0 1 1 4 0 4 4 0 =negative by MLPA and 10th IHW 1 = positive by MPLA and 10th IHW 2 =positive by MLPA and negative by 10th IHW 3 = negative by MLPA andpositive by 10th IHW 4 = not typed by 10th IHW but positive by MLPA

negative by two laboratories and positive typed by one

TABLE 12 Verification of KIR MLPA probe set 1 on 5 PCR-SSP KIR typedsamples. PCR-SSP KIR typed DNA, results of probe set 1. sample 2DL1 2DL22DL3 2DL4 2DL5 2DL5A 2DS1 2DS2 2DS3 2DS4 2DS5 3DL1 3DL2 3DL3 3DS1 2DP13DP1 33_7536 1 0 1 1 1 3 1 2 1 1 1 1 1 1 1 1 1 33_8025 1 0 1 1 0 3 0 2 01 0 1 1 1 0 1 1 33_8037 1 0 1 1 1 3 1 2 0 1 1 1 1 1 1 1 1 33_8588 0 1 01 0 3 0 1 0 1 0 1 1 1 0 0 1 33_9097 1 1 0 1 1 3 1 1 1 0 1 0 1 1 1 1 1 0= negative by MLPA and SSP 1 = positive by MPLA and SSP 2 = positvie byMLPA and negative by SSP 3 = positive by MLPA not typed by SSP

TABLE 13 Verification of KIR MLPA probe set 2 on 11 cell linesKIR-genotyped by the 10^(th) IHW. KIR genotyped Cell lines by the10^(th) IHW, results of probe set2. CODE NAME 2DL1 2DL2 2DL3 2DL4 2DL52DL5A 2DS1 2DS2 2DS3 2DS4 2DS5 3DL1 3DL2 3DL3 3DS1 2DP1 3DP1 231 JVM 1 11 1 0 4 0 1 0 1 2 1 1 4 2 4 4 240 T7507 1 1 1 1 1 4 1 1 1 1 2 1 1 4 1 44 343 OLGA 1 0 1 1 1 4 1 0 0 1 2 1 1 4 1 4 4 423 SAVC 1 0 1 1 0 4 0 0 01 2 1 1 4 2 4 4 712 JBUSH 1 0 1 1 0 4 0 0 0 1 2 1 1 4 2 4 4 723 BM16 1 01 1 0 4 2 0 0 1 2 1 1 4 2 4 4 773 LBUF

1 2 1 3 4 1 1 3 1 1 1 1 4 1 4 4 931 AMALA 1 1 1 1 1 4 1 1 0 1 1 1 1 4 14 4 1042 BM90 1 1 1 1 1 4 1 1 1 1 1 1 1 4 1 4 4 1102 TAB089 1 0 1 1 0 42 0 0 1 2 1 1 4 2 4 4 122 KAS116 1 0 1 1 0 4 2 0 0 1 2 1 1 4 2 4 4 0 =negative by MLPA and 10th IHW 1 = positive by MPLA and 10th IHW 2 =positive by MLPA and negative by 10th IHW 3 = negative by MLPA andpositive by 10th IHW 4 = not typed by 10th IHW but positive by MLPA

negative by two laboratories and positive typed by one

TABLE 14 Verification of KIR MLPA probe set 21 on 5 PCR-SSP KIR typedsamples. PCR-SSP KIR typed patients, results of probe set 2. sample 2DL12DL2 2DL3 2DL4 2DL5 2DL5A 2DS1 2DS2 2DS3 2DS4 2DS5 3DL1 3DL2 3DL3 3DS12DP1 3DP1 33_7536 1 0 1 1 1 4 1 0 1 1 1 1 1 1 1 1 1 33_8025 1 0 1 1 0 40 0 0 1 2 1 1 1 2 1 1 33_8037 1 0 1 1 1 4 1 0 0 1 1 1 1 1 1 1 1 33_85882 1 0 1 0 4 0 1 0 1 2 1 1 1 2 0 3 33_9097 1 1 0 1 1 4 1 1 1 0 1 2 1 1 11 1 0 = negative by MLPA and SSP 1 = positive by MPLA and SSP 2 =positive by MLPA and negative by SSP 3 = negative by MLPA and positiveby SSP 4 = positive by MLPA not typed by SSP

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1-16. (canceled)
 17. A probe or a probe set selected from the probes orprobe sets listed in FIG. 3A, 3B, 3C or 3D, preferably selected from theprobe sets of FIG. 3C or 3D.
 18. Mixture of nucleic acids, wherein saidnucleic acids comprise at least two probes or probe sets according toclaim
 17. 19. A kit for detecting the presence of at least one targetnucleic acid sequence in a sample, comprising a probe or a probe set ora mixture of nucleic acids according to claim
 17. 20. A kit according toclaim 19, wherein said at least one target nucleic acid sequencecomprises a nucleic acid sequence present in a KIR locus.
 21. A kitaccording to claim 19, further comprising a PCR primer set comprising atleast 70%, preferably at least 80%, more preferably at least 85%, morepreferably at least 90%, most preferably at least 95% sequence identityto nucleic acid sequences 5′-GGGTTCCCTAAGGGTTGGA andTCTAGATTGGATCTTGCTGGCAC-3′ or the complements thereof. 22-32. (canceled)